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+title         | COMPETITIVE MICROBIAL AND GEOCHEMICAL PROCESSES CONTROLLING
+blank         | 
+title         |                     THE FATE OF ARSENIC
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+              | 
+              | 
+              | 
+text          |                       A DISSERTATION
+blank         | 
+text          | SUBMITTED TO THE DEPARTMENT OF ENVIRONMENTAL EARTH SYSTEM
+blank         | 
+text          |                           SCIENCES
+blank         | 
+text          |           AND THE COMMITTEE ON GRADUATE STUDIES
+blank         | 
+text          |                   OF STANFORD UNIVERSITY
+blank         | 
+text          |          IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
+blank         | 
+text          |                      FOR THE DEGREE OF
+blank         | 
+text          |                    DOCTOR OF PHILOSOPHY
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+              | 
+              | 
+              | 
+text          |                      Samantha Chi-Yun Ying
+blank         | 
+text          |                           August 2011
+              |               © 2011 by Samantha Chi-Yun Ying. All Rights Reserved.
+              |          Re-distributed by Stanford University under license with the author.
+blank         | 
+              | 
+              | 
+text          |                          This work is licensed under a Creative Commons Attribution-
+              |                          Noncommercial 3.0 United States License.
+              |                          http://creativecommons.org/licenses/by-nc/3.0/us/
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+              | 
+              | 
+              | 
+text          | This dissertation is online at: http://purl.stanford.edu/bg608dx2253
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+              | 
+              | 
+              | 
+meta          |                                           ii
+text          | I certify that I have read this dissertation and that, in my opinion, it is fully adequate
+              | in scope and quality as a dissertation for the degree of Doctor of Philosophy.
+blank         | 
+text          |                                                             Scott Fendorf, Primary Adviser
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+              | 
+              | 
+text          | I certify that I have read this dissertation and that, in my opinion, it is fully adequate
+              | in scope and quality as a dissertation for the degree of Doctor of Philosophy.
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+text          |                                                      Christopher Francis, Primary Adviser
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+              | 
+              | 
+text          | I certify that I have read this dissertation and that, in my opinion, it is fully adequate
+              | in scope and quality as a dissertation for the degree of Doctor of Philosophy.
+blank         | 
+text          |                                                                                  Chad Saltikov
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+              | 
+              | 
+              | 
+text          | Approved for the Stanford University Committee on Graduate Studies.
+              |                                  Patricia J. Gumport, Vice Provost Graduate Education
+blank         | 
+              | 
+              | 
+              | 
+text          | This signature page was generated electronically upon submission of this dissertation in
+              | electronic format. An original signed hard copy of the signature page is on file in
+              | University Archives.
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+              | 
+              | 
+              | 
+meta          |                                           iii
+              | iv
+title         | Abstract
+blank         | 
+text          | Consumption of naturally occurring arsenic (As) in groundwater likely poses the greatest
+              | threat to the most number of humans around the world today. Exposure to high As
+              | concentrations, occasionally up to three orders of magnitude higher than the
+              | recommended limit of 10 µg L-1 set by the World Health Organization, has lead to the
+              | chronic poisoning of millions in South and Southeast Asia.
+              |          Within soil environments, reducing conditions generally promote release of As
+              | into the aqueous phase, while oxidizing conditions favor As immobilization. While many
+              | past studies have examined and illustrated the role of reduction and oxidation processes
+              | individually, there is currently a lack of information on competitive redox or coupled
+              | processes that may occur within soils.      Furthermore, the combined effects of these
+              | chemical processes in soils is complicated by the complex physical structure within
+              | which these reactions take place. Accordingly, this dissertation focuses on defining the
+              | competitive reactions controlling As transformation and partitioning within the physically
+              | and chemically heterogeneous structure of soils.
+              |          Within soils and sediments, redox gradients resulting from mass transfer
+              | limitations lead to competitive reduction-oxidation reactions that drive the fate of As. In
+              | Chapter 2, I investigated As reduction-oxidation dynamics in a diffusively-controlled
+              | system using a Donnan reactor where birnessite and Shewanella sp. ANA-3 are isolated
+              | by a semi-permeable membrane through which As migrates. Initially, As(III) is rapidly
+              | oxidized to As(V) by birnessite; however, a rapid decline in the rate of As(III) oxidation
+              | was observed owing to passivation of the birnessite surface. Modeling and experimental
+              | results show that high [Mn(II)] combined with increasing [CO32-] from microbial
+              | respiration leads to the precipitation of rhodochrosite, which eventually passivates the Mn
+              | oxide surface, inhibiting further As(III) oxidation. These findings show that despite the
+              | initial capacity of birnessite to rapidly oxidize As(III), the synergistic effect of intense
+              | As(V) reduction by microorganisms and the buildup of reactive metabolites capable of
+blank         | 
+meta          |                                                                                           v
+text          | passivating reactive mineral surfaces will produce (bio)geochemical conditions outside of
+              | those based on thermodynamic predictions.
+              |          Mass transfer limitations within soils also promote the formation of competitive
+              | sorption interfaces, where As mobility can be affected by multiple sorbents. Manganese
+              | and Fe oxides are ubiquitous solids in terrestrial systems that have high sorptive
+              | capacities for As. Although numerous studies have characterized the effects of As
+              | adsorption onto Fe and Mn oxides individually, the fate of As within mixed systems
+              | representative of natural environments is unresolved. In Chapter 3, I examined As(III)
+              | oxidation and competitive retention of As on goethite and birnessite by employing the
+              | Donnan reactor. It was found that As(V) is preferentially partitioned onto goethite due to
+              | higher sorption affinity compared to birnessite. Furthermore, reactive transport modeling
+              | demonstrates that the amount of aqueous As available is controlled by the sorption
+              | capacity of the goethite surface, which when saturated, leads to increased aqueous As
+              | concentrations. These findings show that Mn oxides in soils act as a temporary sorbent
+              | of As, but operate primarily as strong oxidants responsible for transformation of As(III)
+              | to As(V), which can then strongly adsorb on, and is ultimately immobilized by, the
+              | surrounding Fe oxide matrix.
+              |          The aggregate-based structure of soils imparts physical heterogeneity that gives
+              | rise to variation in microbial and chemical processes that may influence the speciation
+              | and retention of As. To examine the impact of distributed redox conditions on the fate of
+              | As in aggregated soils, I imposed various redox treatments upon constructed soil
+              | aggregates composed of ferrihydrite- and birnessite-coated sands presorbed with As(V)
+              | and inoculation with Shewanella sp. ANA-3. In Chapter 4, I reveal that diffusion-limited
+              | transport allows reducing conditions to persist in the interior of the aggregate when
+              | aerated treatments are imposed, causing As, Mn, and Fe to migrate from the reduced
+              | aggregate interiors and become immobilized at the aerated exterior region.          Upon
+              | transition to anoxic conditions, pulses of As, Mn and Fe are released into the advecting
+              | solution outside of the aggregate.      These results demonstrate the importance of
+blank         | 
+meta          | vi
+text          | considering redox conditions and the physical complexity of soils in determining the As
+              | dynamics, where redox transitions can either enhance or inhibit As release due to
+              | speciation shifts in both sorbents and sorbates.
+              |          The physical and chemical heterogeneity of soils is accompanied by the great
+              | biological diversity that influences many of the chemical reactions controlling As
+              | transformation. Extensive flooding during monsoon seasons in many regions of South
+              | and Southeast Asia, such as Cambodia, creates anoxic soil conditions that favor anaerobic
+              | microbial metabolic processes, including microbial As(V) respiration. Few studies have
+              | successfully amplified arrA without prior enrichment and factors influencing sequence
+              | diversity are currently unknown. In Chapter 5, amplification of a highly conserved
+              | functional gene encoding dissimilatory As(V) reductase, arrA, was used as a molecular
+              | marker to detect the genetic potential for As(V) respiration in environmental samples. I
+              | demonstrated successful amplification, cloning, and sequencing of 223 novel arrA gene
+              | sequences from Cambodia soils without prior enrichment/stimulation, collectively
+              | forming a clade that is phylogenetically distinct from existing sequences available.
+              | Application of permutational MANOVA demonstrates that As and depth variables are
+              | most strongly associated with variations in arrA sequences. These findings demonstrate
+              | the potential for using biogeochemically and ecologically relevant functional genes to
+              | understand operative geochemical processes.
+              |          In sum, strong oxidants of As, such as Mn oxides, may impede As transport by
+              | transforming As(III) to the less mobile As(V) form; however, aqueous As concentrations
+              | are ultimately controlled by the availability of Fe oxide sorption sites. Furthermore,
+              | diffusion limited transport induces the formation of reducing centers within soil
+              | aggregates even in the presence of oxygen. Reduced Fe that diffuses from the aggregate
+              | interior forms a protective Fe oxide barrier upon contact with oxygenated advective flow
+              | exterior of the aggregate, which adsorbs As from the interior, inhibiting its release to the
+              | advecting solution. However, elimination of oxygen from the advecting solution causes a
+              | pulse of As to be released upon reductive dissolution of the Fe oxide rind. Overall, the
+blank         | 
+meta          |                                                                                          vii
+text          | research presented here reveals the importance of considering competitive chemical and
+              | biological reactions that arise within mass transfer limited environment when evaluating
+              | the fate and transport of As within soils.
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+              | 
+              | 
+              | 
+meta          | viii
+              | Dedication
+blank         | 
+text          |                          For my Wai-Wai
+blank         | 
+text          |                        For     ∘∘子子
+blank         | 
+text          |        and past, present, and future members of the Ying Clan
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+              | 
+              | 
+              | 
+meta          |                                                                 ix
+              | x
+title         | Acknowledgements
+blank         | 
+text          | First and foremost I must thank Scott, who has not only been an exceptional PhD advisor,
+              | but also an amazing mentor for many things in life. I always appreciated how Scott
+              | placed so much of his effort into making our lives whole, recognizing that a student’s
+              | productivity directly linked with our overall happiness. Whenever I doubted my own
+              | abilities, Scott always had the right words to say and always went far and beyond to make
+              | things work. Raising a baby during grad school can really turn your life upside down and
+              | test all your limits, but Scott aided me in figuring out a brand new schedule to my day
+              | along with revamping my emotional approach to life in general, which taught me how to
+              | enjoy every moment with a perpetually positive attitude. I couldn’t be more grateful for
+              | all the opportunities Scott has provided me over these phenomenally long seven years of
+              | growth--he is a true inspiration.
+              |          I must also thank my co-advisor Chris Francis and committee members Ali
+              | Boehm and Chad Saltikov for their encouraging attitudes and continuous support. I also
+              | thank my undergraduate advisor, Mike Goodchild for providing me guidance through
+              | numerous internship opportunities, fostering my love for research, and eventually guiding
+              | me toward my Stanford career.
+              |          I thank past and present Fendorfians, Kate, Colleen, MattyP, MattGV, Matt
+              | Lappe, Hanne, Jessica, Jessi, Jason Stuckey, Mike, Debra, Angelia, for making the lab
+              | such a fun place. I thank Elise for being one of the world’s funniest people and greatest
+              | officemates and Sarah for being the best intern I could’ve ever had. Yoko, who I
+              | consider one of my “thesis co-authors,” has been like a sister to me throughout these
+              | seven years; we started together in our window-less office and we’re still together almost
+              | everyday. She spent countless days in the lab with me and I couldn’t thank her more for
+              | her mentoring and companionship. Together we made it through as Moms!
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+              | 
+              | 
+meta          |                                                                                         xi
+text          |          I would have 90% less data if it were not for Guangchao and his expertise on all
+              | our analytical machines. I also thank Sam Webb, Matthew Marcus for their help with
+              | synchrotron data and Lynsey and Robin for their administrative support.
+              |          I also thank Ben Kocar, my other “thesis co-author.” Ben is the most amazing
+              | hands-on scientist I know and definitely one of the most intelligent. He was at many
+              | times my “mini-advisor” who patiently guided me through all the stupid questions, but
+              | also kept me on track with the big picture, and always had faith in me. I cherish the times
+              | we had in Cambodia and the friendship we’ll have for life. And of course I have to thank
+              | him for being a phenomenal father to our daughter, Wai Wai, the biggest, most wonderful
+              | accomplishment that could have come out during our PhDs!
+              |          I thank Pie and Kevan for bringing me ice cream when I needed it, cheering me
+              | up when I was down and being two of the most dependable people I’ve ever known. I
+              | thank Jason for the endless hours of fantastic conversation, and being a fun, wonderful,
+              | hilarious friend. Beck, Alys, Jfer, Suse, Sony, thank you for always being the best. I
+              | must thank Carmen and PJ for changing my life, showing me how to live again, and
+              | being my life support at all times. Thank you for all the little things that make my
+              | everyday so fantastic; without you, finishing my PhD would have been nearly impossible.
+              |          Most of all I thank Pa and Ma, my idols and perpetual guidance. Pa taught me
+              | and my sister how to be interested in everything and bends over backwards to help us
+              | accomplish anything we ever wanted. Ma taught me to be strong, to never regret, and
+              | always live life to the fullest. Gi, my obligatory bestfriend, who constantly reminds me
+              | there’s more to life than work and always makes me laugh until I cry. Thank you Tuba
+              | for taking care of Wai Wai like one of your own and bringing my adorable nephews
+              | Sandwich, Lolly, and Momo into my life all during my seven years at Stanford!
+              |          Last, but definitely far from the least, my little lady, Wai Wai. I thank her for
+              | being the best daughter and buddy anyone could ever imagine and always being the
+              | highlight of my day. I thank her for her continuous silliness, her craziness, and her love.
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+              | 
+              | 
+              | 
+meta          |                                                                                         xii
+title         | Table of Contents
+text          | Abstract ................................................................................................................................v!
+              | Dedication .......................................................................................................................... ix!
+              | Acknowledgements............................................................................................................ xi!
+              | Chapter 1: Introduction ........................................................................................................1!
+              |      1.1 History of Arsenic Exposure....................................................................................1!
+              |      1.2 Mechanisms of Arsenic Toxicity .............................................................................2!
+              |      1.3 Impact of Redox Processes on As mobility .............................................................2!
+              |      1.4 Impact of As(V) and Fe(III) Reduction on Fate of As.............................................3!
+              |      1.5 Microbial As(V) Reduction .....................................................................................4!
+              |      1.6 Arsenic(III) oxidation ..............................................................................................7!
+              |      1.7 Arsenic sorption dynamics in soil environments .....................................................9!
+              |      1.8 Physical heterogeneity of soils and sediments.........................................................9!
+              |      1.9 Motivation of Research..........................................................................................11!
+              |      1.10 Scope of Research................................................................................................11!
+              |      1.11 References............................................................................................................15!
+              | Chapter 2: Competitive Microbially and Mn oxide Mediated Redox Processes
+              |      Controlling Arsenic Speciation and Partitioning .........................................................23!
+              |      2.1 Abstract ..................................................................................................................23!
+              |      2.2 Introduction............................................................................................................24!
+              |      2.3 Materials and Methods...........................................................................................27!
+              |            2.3.1 MnO2 synthesis and characterization............................................................27!
+              |            2.3.2 Abiotic As(III)/birnessite incubations...........................................................28!
+              |            2.3.3 Shewanella sp. ANA-3 preparation. .............................................................28!
+              |            2.3.4 Donnan Cell and Experiments. .....................................................................29!
+              |            2.3.5 Aqueous phase analysis. ...............................................................................30!
+              |            2.3.6 Solid phase analysis. .....................................................................................30!
+blank         | 
+meta          |                                                                                                                                      xiii
+text          |         2.3.7 Reactive Transport Modeling. ......................................................................32!
+              |    2.4 Results....................................................................................................................32!
+              |    2.5 Discussion ..............................................................................................................37!
+              |         2.5.1 Rate Controlling Processes. ..........................................................................37!
+              |         2.5.2 Competing Redox Processes and Operative Reaction Network. ..................38!
+              |    2.6 Acknowledgements...............................................................................................43!
+              |    2.7 References.............................................................................................................43!
+              | Chapter 3: Competitive Adsorption of Arsenic Between Goethite and Birnessite............47!
+              |    3.1 Abstract ..................................................................................................................47!
+              |    3.2 Introduction............................................................................................................48!
+              |    3.3 Material and Methods ............................................................................................51!
+              |         3.3.1 Birnessite synthesis and goethite characterization........................................51!
+              |         3.3.2 Donnan experiment conditions. ....................................................................51!
+              |         3.3.3 Adsorption isotherms. ...................................................................................52!
+              |         3.3.4 Aqueous phase analysis. ...............................................................................53!
+              |         3.3.5 Solid phase analysis. .....................................................................................53!
+              |         3.3.6 Reactive transport modeling. ........................................................................55!
+              |    3.4 Results....................................................................................................................55!
+              |         3.4.1 Aqueous As dynamics within Donnan reactor..............................................55!
+              |         3.4.2 Arsenic sorption onto goethite and birnessite...............................................57!
+              |         3.4.3 Control experiments and reactive transport model calibration. ....................59!
+              |    3.5 Discussion ..............................................................................................................60!
+              |    3.6 Conclusions...........................................................................................................64!
+              |    3.7 Acknowledgements................................................................................................65!
+              |    3.8 References.............................................................................................................65!
+              | Chapter 4: Distributed microbially- and chemically-mediated redox processes
+              |    controlling arsenic dynamics within Mn-/Fe-oxide constructed aggregates ...............69!
+              |    4.1 Abstract ..................................................................................................................69!
+              |    4.2 Introduction............................................................................................................70!
+blank         | 
+meta          |                                                                                                                                xiv
+text          |      4.3 Materials and Methods...........................................................................................72!
+              |           4.3.1 Aggregate construction and reactor setup.....................................................72!
+              |           4.3.2 Flow-through reactor experimental procedure..............................................73!
+              |           4.3.3 Aqueous phase analysis. ...............................................................................74!
+              |           4.3.4 Solid phase analyses. ....................................................................................74!
+              |      4.4 Results....................................................................................................................76!
+              |           4.4.1 Aqueous phase results from aggregate reactors............................................76!
+              |           4.4.2 Solid phases analysis.....................................................................................82!
+              |      4.5 Discussion ..............................................................................................................87!
+              |      4.6 Conclusions............................................................................................................90!
+              |      4.7 Acknowledgements................................................................................................91!
+              |      4.8 References..............................................................................................................91!
+              | Chapter 5: Analysis of Arsenic(V)-reducing microbial community structure and
+              |      environmental influences using multivariate statistics ................................................95!
+              |      5.1 Abstract ..................................................................................................................95!
+              |      5.2 Introduction............................................................................................................96!
+              |      5.3 Materials and Methods...........................................................................................98!
+              |           5.3.1 Site description and soil sampling. ...............................................................98!
+              |           5.3.2 Lysimeter installation and water collection. .................................................98!
+              |           5.3.3 Genomic DNA extraction and PCR amplification of arsenate reductase
+              |                 (arrA) genes. ....................................................................................................99!
+              |           5.3.5 Quality control and phylogenetic analysis of arrA gene sequences. ..........100!
+              |           5.3.6 Multivariate analysis of arrA genes and environmental factors. ................102!
+              |      5.4 Results and Discussion ........................................................................................102!
+              |           5.4.1 Phylogenetic analysis of arrA genes in Cambodian sediments. .................102!
+              |           5.4.2 Permutational MANOVA analysis. ............................................................106!
+              |      5.5 References............................................................................................................107!
+              | Chapter 6: Summary ........................................................................................................111!
+              |      6.1 References............................................................................................................115!
+blank         | 
+meta          |                                                                                                                                   xv
+text          | APPENDIX A: Supporting information for Chapter 2....................................................117!
+              |     A.1 Reactive Transport Modeling using MIN3P.......................................................117!
+              |     A.2 Carbonate calculations ........................................................................................120!
+              |     A.3 Saturation index calculations ..............................................................................120!
+              |     A.4 Diffusion coefficient calculations .......................................................................121!
+              |     A.5 References cited in Appendix A ........................................................................121!
+              | APPENDIX B: Supporting Information for Chapter 3....................................................125!
+              |     B.1 Reactive Transport Modeling using MIN3P .......................................................125!
+              |     B.2 Diffusion coefficient calculations .......................................................................126!
+              |     B.3 References cited in Appendix B.........................................................................129!
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                                                      xvi
+title         | List of tables
+blank         | 
+text          | Number                                                                                                                              Page
+              | Table 2.1 Reaction network used within the reactive transport model to describe arsenic
+              |             dynamics..................................................................................................................... 31!
+              | Table 3.1 Reactions and parameters considered in reactive transport modeling using MIN3P. ... 54!
+              | Table 4.1 Solid phase characteristics of aggregate sections .......................................................... 83!
+              | Table 4.2 Solid phase As and Fe speciation in aggregate sections ................................................ 85!
+              | Table 5.1 Concentration of aqueous constituents in porewater samples collected in Kien
+              |             Svay, Kandal Province, Cambodia ............................................................................. 99!
+              | Table 5.2 Weighted UniFrac distance matrix used in PERMANOVA analysis. Definition of
+              |             sample number with corresponding sample information is provided in Table 1. .... 102!
+              | Table 5.3 Permutational MANOVA results using adonis function of vegan package. ............... 106!
+              | Table A.1 Rate expressions for reactions used in simulation. ..................................................... 118!
+              | Table A.2 Kinetic and thermodynamic values used in reactive transport simulations. ............... 119!
+              | Table B.1 Rate expressions for reaction used in simulation ........................................................ 126!
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                                                                       xvii
+              | xviii
+title         | List of figures
+blank         | 
+text          | Number                                                                                                                           Page
+              | Figure 2.1 Schematic illustration of diffusion limited transport and formation of redox
+              |             gradients in soil aggregates (A) and schematic overview of arsenic cycling
+              |             between a suboxic zone (containing birnessite) and an anoxic zone (containing
+              |             dissimilatory metal reducing bacteria, Shewanella) as determined by Donnan cell
+              |             experiment (B)............................................................................................................ 26!
+              | Figure 2.2 Donnan reactor dimensions and experimental set up. .................................................. 29!
+              | Figure 2.3 Aqueous arsenic concentrations as a function of time in (A) birnessite and (B)
+              |             Shewanella chambers, and (C) solid-phase As concentrations in birnessite. Total
+              |             aqueous arsenic (black dots), As(V)aq (black squares), and As(III)aq (white
+              |             squares) are shown. Dotted lines represent model results. ........................................ 34!
+              | Figure 2.4 Total aqueous Mn concentrations in Shewanella and birnessite chambers (squares
+              |             and white circles, respectively, right axis) and rhodochrosite saturation index
+              |             (black circles, left axis) are shown in (A). Low angle x-ray diffraction patterns
+              |             for birnessite harvested from Donnan reactor after experiment termination (B
+              |             top), birnessite incubated with 1.6 mM As(III) (B middle), and birnessite with no
+              |             additions (B bottom) are also shown. Major peaks are labeled as birnessite, B,
+              |             and rhodochrosite, R................................................................................................... 36!
+              | Figure 2.5 Simulated aqueous As concentrations in Shewanella and birnessite chambers when
+              |             MnCO3 inhibition term is removed in the presence of 3 mM lactate (top) and
+              |             when lactate concentrations are decreased to 0.03 mM (bottom) in reactive
+              |             transport simulations. Total As (black and white dots), As(V) (black squares),
+              |             and As(III) (white squares) concentrations are shown. .............................................. 39!
+              | Figure 2.6 Simulated As (aq) concentrations in Shewanella (left panel) and birnessite(right
+              |             panel) chambers as lactate concentrations were decreased from 1 mM to 0.03
+              |             mM in reactive transport simulations. In the presence of 0.3 mM lactate
+              |             (middle), the electron donor becomes limiting at approximately 70 h, as shown
+              |             by the decreasing As(III) concentrations in the Shewanella chamber indicating a
+              |             lowered As(V) reduction rate. At approximately 110 h, the rate of As(V)
+              |             reduction decreases below the As(III) oxidation rate within the birnessite
+              |             chamber. Although As(III) oxidation by birnessite is inhibited, it is still
+              |             operative, and therefore As(V) concentrations begin to increase once again. At
+              |             0.03 mM lactate concentrations (bottom), As(V) reduction by Shewanella cannot
+              |             be 9 performed and, therefore, carbonate and Mn(II) concentrations do not reach
+              |             concentrations great enough to precipitate rhodochrosite, allowing uninhibited
+              |             oxidation of injected As(III). Symbols represent total Asaq (dots), As(V)aq (black
+              |             squares), As(III)aq (open squares). .............................................................................. 42!
+              | Figure 3.1 Competitive sorption reactions within mass transfer limited soil systems, such as
+              |             an rhizosphere aggregates composed of a mixture of Mn oxides along root-zones
+blank         | 
+              | 
+meta          |                                                                                                                                     xix
+text          |               within an Fe oxide matrix, is simulated and quantified using the Donnan reactor
+              |               (bottom panel)............................................................................................................. 49!
+              | Figure   3.2 Aqueous arsenic concentrations in goethite chamber (right panel) and birnessite
+              |               chamber (left panel) when 480 µM (top) and 40 µM (bottom) As(III) is injected
+              |               into the reactor. Total aqueous arsenic (black dots), As(V)aq (white squares), and
+              |               As(III)aq (black squares) are shown. Dotted line represents model results for
+              |               aqueous As(V) and dashed line for aqueous As(III). ................................................. 56!
+              | Figure    3.3 Solid phase arsenic concentrations in goethite (white circles) and birnessite
+              |               chamber (black circles) when 480 µM (A) and 40 µM (B) As(III) is injected into
+              |               the reactor. Dotted line represents model results for sorption onto birnessite and
+              |               dashed line for sorption onto goethite. ....................................................................... 57!
+              | Figure   3.4 Fraction of As(V) (white symbols) and As(III) (black symbols) adsorbed on
+              |               goethite as determined by XANES analysis when 480 µM (circles) or 40 µM
+              |               As(III) (squares) was added into Donnan reactor (A). Total solid phase Mn
+              |               concentrations in goethite digestions when 480 µM (black circles) or 40 µM
+              |               As(III) (white triangles) is added into Donnan reactor (B). ....................................... 58!
+              | Figure    3.5 Arsenic(III) input concentration in As(III) versus sorbed As on birnessite
+              |               As(III)/birnessite incubations ..................................................................................... 59!
+              | Figure   3.6 Predictive modeling results of aqueous As with 0.4, 4, 10, and 20 µM As input in
+              |               the birnessite chamber (A) and goethite chamber (B) with insets showing
+              |               expanded regions. As(III) (dashed lines), As(V) (solid lines) are shown. (C)
+              |               Predictive modeling results of As adsorbed onto birnessite with 20, 10, 4, and 0.4
+              |               µM of As(III) input. (D) Predictive modeling results of As adsorbed onto
+              |               goethite with 20, 10, 4, and 0.4 µM of As(III) input. ................................................. 61!
+              | Figure   3.7 Predictive model simulations of As concentrations in the aqueous phase and solid
+              |               phase in birnessite chamber (A and C respectively), and aqueous and solid phase
+              |               in goethite chamber (B and D respectively) at 1:10, 1:50, 1:100 goethite to
+              |               birnessite ratios in the presence of 40 µM As(III). Arsenic(III) is instantly
+              |               oxidized. Aqueous As(V) (solid lines) and aqueous As(III) (dashed lines) are
+              |               shown. Lines have multiple labels if data overlaps. .................................................. 63!
+              | Figure   4.1 Dissolved As (A) and Fe and Mn (B) in effluent from aerated reactor. Dissolved
+              |               total As (black dots), As(III) (open squares), and As(V) (black squares), Fe
+              |               (white circles), and Mn (black circles) are shown...................................................... 77!
+              | Figure   4.2 Dissolved As (A) and Fe and Mn (B) in effluent from anoxic reactor. Dissolved
+              |               total As (black dots), As(III) (open squares), and As(V) (black squares), Fe
+              |               (white circles), and Mn (black circles) are shown...................................................... 78!
+              | Figure   4.3 Dissolved As (A) and Fe and Mn (B) in effluent from anoxic-to-aerated transition
+              |               reactor. Dissolved total As (black dots), As(III) (open squares), and As(V)
+              |               (black squares), Fe (white circles), and Mn (black circles) are shown. ..................... 80!
+              | Figure   4.4 Dissolved As (A) and Fe and Mn (B) in effluent from aerated-to-anoxic transition
+              |               reactor. Dissolved total As (black dots), As(III) (open squares), and As(V)
+              |               (black squares), Fe (white circles), and Mn (black circles) are shown. ..................... 81!
+              | Figure   4.5 Fraction of final concentration relative to initial for mass of As (gray), Fe (black),
+              |               and Mn (white) in the exterior (E), mid (M), and interior (I) section for aerated
+              |               (A), anoxic (B), aerated-to-anoxic transitioned (C), and anoxic-to-aerated
+              |               transitioned (D) aggregates......................................................................................... 82!
+blank         | 
+              | 
+meta          |                                                                                                                                         xx
+text          | Figure 4.6 X-ray fluorescence mapping of Fe and Mn (top panel 1) and As(III) and As(V)
+              |             (bottom panel 2) in (A) aerated, (B) anoxic, (C) anoxic-to-aerated, (D) aerated-
+              |             to-anoxic aggregates. .................................................................................................. 84!
+              | Figure 4.7 As species distribution for aggregate cross section determined by As !-XANES
+              |             analysis from exterior (E), mid (M), to interior (I) sections of (A) aerated, (B)
+              |             anoxic, (C) aerated-to-anoxic, (D) anoxic-to-aerated................................................. 86!
+              | Figure 5.1 Maximum likelihood phylogram of available ArrA sequences. Sequences were
+              |             required to be at least 190 amino acids in length for inclusion in analysis.
+              |             Sequences obtained from two previous environmental surveys (Song et al. (12);
+              |             Hery et al. (13)) are also included. Tips (phylotypes) are labeled according to
+              |             their source site and depth, the previous survey, or with an isolate name,
+              |             respectively. Multiple symbols at the same tip indicate that the same phylotype
+              |             was observed in multiple samples. The number inside the symbol indicates the
+              |             total number of individuals observed. ...................................................................... 104!
+              | Figure 5.2 Expanded representation of the Cambodia-specific clade (this study)....................... 105!
+              | Figure B.1 Diffusion of 480 !M As(III) (A) and As(V) (B) through semi-permeable
+              |             polycarbonate membrane.                Closed circles and opened circles indicate
+              |             concentration of As in the input chamber and diffusion chambers, respectively..... 127!
+              | Figure B.2 Diffusion controlled sorption of As(III) or (V) on birnessite or goethite. One of
+              |             the oxides was placed in a reaction chamber in As was then injected in the
+              |             opposing chamber; the temporal changes in (top four panels) and sorbed (bottom
+              |             four panel) As concentrations were then followed................................................... 128!
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                                                                   xxi
+title         | Chapter 1: Introduction
+blank         | 
+title         | 1.1 History of Arsenic Exposure
+text          |          Exposure to arsenic (As), a naturally occurring toxic metalloid, has lead to the
+              | illness and death of millions of people over many centuries. Scheele’s green, a greenish
+              | yellow pigment composed of cupric hydrogen arsenite, was invented in 1775 by Swedish
+              | pharmaceutical chemist Carl Wilhelm Scheele, and was used to pigment many everyday
+              | household items including paints, food-dye, candle-wax, and fabric (1). Illness caused by
+              | exposure to Scheele’s green through consumption or inhalation (as arsine gas, AsH3) and
+              | its blackening when subjected to hydrogen sulfide eventually lead to its replacement with
+              | cobalt green (1). Similarly, many other anthropogenic applications and activities have
+              | lead to human exposure to toxic concentrations of As, including its use as an additive in
+              | pesticides, herbicides, and feedstock (2), and its release during mining (3), metal smelting
+              | (4), and coal combustion (5).      According to the Total Diet Market Basket Study
+              | conducted by the FDA for the years 1991-1997, total daily intake of As in the U.S, is less
+              | than 20 µg assuming consumption of 2000 mL of drinking water containing a maximum
+              | of 5 µg L-1 of As and approximately 10 µg from food intake (6).
+              |          Consumption of naturally occurring As in groundwater likely poses the greatest
+              | threat to the most number of humans around the world today. Exposure to high As
+              | concentrations, occasionally up to three orders of magnitude higher than the
+              | recommended limit of 10 µg L-1 set by the World Health Organization (7), has lead to the
+              | chronic poisoning of millions in South and Southeast Asia (8, 9), many of whom are
+              | exhibiting a wide range of health problems including skin lesions due to
+              | hyperpigmentation and hyperkertosis (10, 11) and terminal cancers (8, 12, 13).
+title         | 1.2 Mechanisms of Arsenic Toxicity
+text          |          Arsenic exists in both inorganic and organic forms with varying oxidation states
+              | (-3, 0, +3, and +5), where As(III) compounds have more potent toxic properties than
+              | those of As(V) compounds as determined by LD50 values of arsenicals in laboratory
+              | animals (14). The lethal range of inorganic As for a human adult is estimated to be 1 to3
+              | mg As kg-1 (15). In humans and many other mammals, As(V) is rapidly reduced to
+              | As(III) in the liver by glutathione-S-transferase (16), which can occur non-enzymatically
+              | in the presence of glutathione (GSH), followed by oxidative methylation to monomethyl
+              | arsenic (MMA) or dimethyl arsenic (DMA) (17).
+              |          Arsenic(III)   readily    reacts   with   thiol-containing    molecules     including
+              | multifunctional enzymes which leads to the inhibition of key cellular functions.
+              | Arsenic(III) has a high affinity for dithiols, such as lipoic acid, a cofactor required for the
+              | enzymatic functioning of pyruvate dehydrogenase (PDH). PDH is responsible for the
+              | oxidation of pyruvate to acetyl-CoA, the activated precursor for the citric acid cycle
+              | where reducing equivalents (e.g. NADH) are produced to fuel the production of ATP
+              | through electron transport.
+              |          Arsenic(V) is a chemical analog of phosphate, having similar structure and
+              | chemical properties, allowing As(V) to replace phosphate in many cellular processes.
+              | For example, reaction of As(V) with glucose can form glucose-6-arsenate, in replacement
+              | of glucose-6-phosphate (G6P), as a substrate for G6P dehydrogenase which inhibits
+              | hexokinase activity (required to direct glucose toward glycolysis or glycogenesis
+              | pathways).    Aside from uncoupling glycolytic pathways, As(V) can also replace
+              | phosphate during substrate level phosphorylation. ATP is formed during glycolysis in
+              | the presence of phosphate, but not in the presence of arsenate (18).
+blank         | 
+              | 
+title         | 1.3 Impact of Redox Processes on As mobility
+text          |          The degree of As mobility in soils and sediments is governed, in large part, by
+              | redox transformations of As and Fe species as seen in soils and sediments of South and
+              | Southeast Asia. Cyclic redox conditions in Bangladesh and Cambodia have resulted in
+meta          |                                                                                   2
+text          | the release of As from surface sediments followed by eventual transport into aquifers
+              | below (19, 20). Arsenic release from As-bearing sulfur minerals during rock weathering
+              | leads to their repartitioning on Fe and Mn oxides.        After sediment transport and
+              | deposition, the onset of reducing conditions with burial results in As release from the
+              | oxides, with As, Fe, and Mn entering the aqueous phase. Once As partitions into the
+              | aqueous, it is transported through sediments by a combination of advective flow paths
+              | between aggregates and diffusion-dominated flow within aggregates. The remainder of
+              | this chapter serves as an introduction to many biogeochemical mechanisms controlling
+              | As mobilization in soils and sediments; in particular, oxidation and reduction processes
+              | driven by biotic and abiotic forces, sorption mechanisms, and the physical heterogeneity
+              | of soil.
+blank         | 
+              | 
+title         | 1.4 Impact of As(V) and Fe(III) Reduction on Fate of As
+text          |            Arsenic(III) and As(V) are the dominant As oxidation states in soils and
+              | sediments, where As(V), typically present as HxAsO4x-3, adsorbs to a wide-range of
+              | minerals including iron and aluminum (hydr)oxides and aluminosilicates minerals;
+              | As(III), by contrast, is usually present as the neutral H3AsO3 species in non-sulfidic
+              | environments and preferentially adsorbs to iron (hydr)oxides (21).
+              |            Both As(V) and As(III) are able to adsorb strongly onto Fe oxides (here
+              | collectively referring to oxyhydroxides, hydroxides, and oxides), forming bidentate-
+              | binuclear, inner-sphere complexes (22). Sorption processes of As on Fe oxides control
+              | the availability of As in many environments including acid mine drainage, marine
+              | systems, and sediments (2). The effectiveness of Fe oxides for As sorption has been
+              | applied in adsorptive treatment and development of other As remediation technologies
+              | used in water treatment processes (23-25). Although Fe oxides are effective adsorbents
+              | of As, labile, outer-sphere complexes may comprise a large portion of surface population
+              | (26-28), which can be mobilized under flow conditions.          Arsenic(III) binds more
+              | extensively on Fe oxide surfaces than As(V), but is more weakly bound compared to
+blank         | 
+              | 
+              | 
+meta          |                                                                                       3
+text          | As(V) as indicated by its more rapid and extensive desorption under advective flow (29-
+              | 31).
+              |          Reduction of Fe(III) (hydr)oxides can also lead to As mobilization through
+              | reductive dissolution and mineral transformation. Transformation of ferrihydrite, a poorly
+              | crystalline Fe(III) oxide with high surface area and sorption capacity for both As(V) and
+              | As(III), to secondary minerals with lower surface area, such as magnetite and goethite
+              | (32), can lead to desorption (33). However, the effects of Fe oxide transformation under
+              | reducing conditions is complex and does not result in As release exclusively.
+              | Interestingly, secondary precipitation of magnetite during microbial respiration of
+              | ferrihydrite has been shown to temporarily sequester As(III) (29), followed by gradual
+              | release as Fe oxide transformation ceases and reductive dissolution proceeds (34).
+              |          To elucidate the mechanisms involved in this dual stage process the relative role
+              | of As(V) and Fe(III) oxide reduction in As desorption under hydrodynamic conditions
+              | was characterized by inoculating Shewanella sp. ANA-3, a well-characterized model
+              | As(V)-respiring bacterium isolated from Eel Pond in Woods Hole, Massachusetts, (8)
+              | capable of Fe(III) and As(V) reduction, in columns containing ferrihydrite- or hematite-
+              | coated sands presorbed with As(V). Furthermore, mutant strains of Shewanella sp.
+              | ANA-3 incapable of As(V) reduction or Fe(III) reduction were used to isolate the relative
+              | effect of the two processes. The greatest amount of As was desorbed in the absence of
+              | Fe(III) reduction, where formation of magnetite was inhibited, preventing sequestration
+              | of As(III), while both Fe(III) reduction only and Fe(III)/As(V) reduction treatments
+              | resulted in temporary As immobilization. Masue-Slowey et al. (35) observed similar As
+              | sequestration effects within the anaerobic center of synthetic aggregates composed of
+              | As(V)-sorbed ferrihydrite coated sands and As(V)-reducing bacteria under aerated
+              | treatment.
+blank         | 
+              | 
+title         | 1.5 Microbial As(V) Reduction
+text          |          Reduction of As(V) in natural environments is often mediated by microbes,
+              | where organic carbon amended to sediment from West Bengal have been shown to
+blank         | 
+meta          |                                                                                         4
+text          | enhance As(V) reduction (36). Microbes are able to reduce As(V) through detoxification
+              | or As(V) respiration under anaerobic conditions. Arsenic detoxification in bacteria is
+              | mediated by the ars system, which has been thoroughly characterized in E. coli.
+              | Arsenic(V) is taken up through phosphate transporters (e.g. Pit system) (37) while As(III)
+              | uptake is likely mediated by GlpF, a glycerol facilitator in the aquaporin superfamily (38)
+              | which may mistake As(OH)3 as an inorganic equivalent of glycerol. Arsenic(V) uptake is
+              | followed by reduction to As(III) by arsenate reductase, ArsC, encoded by arsC gene, the
+              | homolog of which is found in many bacteria. Arsenic(V) is reduced in a two-step process
+              | by glutaredoxin and GSH, producing an As(III) intermediate bound to a cysteine residue
+              | that is hydrolyzed to release As(III). Arsenic(III) produced from the reduction of As(V)
+              | by ArsC or by uptake through aquaporins is removed from the cytosol through either an
+              | arsenite carrier protein or an arsenite-translocation ATPase (39). In most bacteria, an
+              | ATPase encoded by arsA is coupled with a cytoplasmic membrane efflux pump, ArsB.
+              | Regulation of the ars operon is facilitated by ArsR and ArsD, which controls the basal
+              | level (Kd of 0.33 µM As(III)) and the maximal level (Kd of 60 µM As(III)) of operon
+              | expression, respectively (40), to prevent overexpression of ArsB.
+              |          Under anaerobic conditions, microorganisms can couple the oxidation of a wide-
+              | range of organic compounds (e.g. H2, lactate, acetate), to the reduction of As(V) during
+              | respiration. Dissimilatory As(V)-reducing prokaryotes (DARPs) have been isolated from
+              | a wide-range of environments (41) and are physiologically and phylogenetically diverse
+              | with representatives from !-, "-, and #-Proteobacteria, low G+C gram positive bacteria
+              | (Firmicutes), Eubacteria, and Crenarchaea (41)
+              |          Although the ability to respire upon As(V) is wide-spread among many phyla,
+              | no obligate As(V)-reducing microorganisms have been isolated, and instead, many are
+              | capable of respiring upon a large spectrum of terminal electron acceptors such as Mn(IV),
+              | Fe(III), Se(V), SO42-, and NO3-. Whereas the ars system is expressed under both aerobic
+              | and anaerobic conditions, the arr operon that encodes proteins necessary for As(V)
+              | respiration is expressed only under anaerobic conditions (42). Both systems are induced
+              | by As(III); however, the arr system is activated at much lower concentrations (>100 nM)
+              | than ars system (>100 µM) (42). The arr operon encodes for ArrA, a molybdenum-
+meta          |                                                                                          5
+text          | containing enzyme and ArrB, an enzyme containing Fe-S clusters, both resembling
+              | proteins within the dimethyl sulfoxide (DMSO) reductase family (43). Arsenic(V) first
+              | binds to ArrA in the periplasm, and is then reduced to As(III) with electrons delivered by
+              | ArrB provided by c-type cytochromes during electron transport (43).
+              |          The arrA gene is well conserved among phylogenetically diverse As(V)-
+              | respiring bacterial isolates, making it possible to design primers to probe for arrA
+              | presence using PCR methods (44-49). Divergences in the arrA sequences from different
+              | locations may be inferred as variations in functionality between environments; hence,
+              | specific environmental conditions may house a distinct clade of As(V)-respiring microbes
+              | detectable through phylogenetic analysis. Few studies have examined the diversity of
+              | arrA directly in the environment (44, 46, 47, 49), and even fewer so have focused on their
+              | presence in Southeast Asia where As(V) reduction processes have great impact on human
+              | health issues.
+              |          Phylogenetic analysis of arrA sequences from two Soda Lakes, Searles and
+              | Mono Lake, containing As concentrations as high as 3.9 mM, showed unique ArrA
+              | phylotypes exist in both lakes, which could be explained by horizontal gene transfer
+              | between the two environments or the existence of unique populations of dissimilatory
+              | As(V)-reducing microorganisms within each lake (46).          Similarly, arrA sequences
+              | acquired from sediments in West Bengal (49) and Chesapeake Bay (47) appear to form
+              | distinct phylogenetic clusters, implying distinct populations of As(V)-respiring microbes;
+              | however, inappropriate methods used for phylogenetic analysis (i.e. neighbor joining
+              | method) may provide results that are misleading regarding the relationship between arrA
+              | communities.     Furthermore, many existing partial arrA sequences available have
+              | insufficient length (e.g. products amplified with primers provided in Malasarn et al are <
+              | 200 bp) or have been amplified from amended samples such as incubation experiments.
+              | To date, only one studies has explore the diversity of arrA in Southeast Asia (Lear et al.,
+              | 2007); however their findings are inconclusive regarding characterizing the phylogeny of
+              | the native As(V)-respiring community due to small dataset (i.e. only 10 sequences were
+              | acquired) and products were amplified from incubation experiments amended with As(V)
+blank         | 
+              | 
+meta          |                                                                                          6
+text          | and acetate rather than untreated sediments. Further characterization of the functional
+              | gene responsible for encoding As(V) reduction in the environment is warranted.
+blank         | 
+              | 
+title         | 1.6 Arsenic(III) oxidation
+text          |          While reductive processes promote As desorption, oxidative processes can
+              | immobilize As correspondingly through sorption on oxide surfaces formed via oxidative
+              | precipitation of Fe(II) and Mn(II), and oxidation of As(III) to the more strongly sorbing,
+              | less toxic As(V) form. Arsenic(III) oxidation can be catalyzed enzymatically by both
+              | heterotrophic and chemoautotrophic microbes as a detoxification mechanism (50) and for
+              | growth, (51), respectively. Additionally, As oxidation can also proceed chemically
+              | through electron transfer to Mn oxides (52-55). Oxidation of Mn(II) released from the
+              | weathering of igneous and metamorphic rocks produces a variety of Mn(III), Mn(IV),
+              | and mixed valence Mn oxides in the environment that thus may contribute to the abiotic
+              | oxidation of As in soils and sediments.
+              |          Whereas abiotic oxidation of Fe(II) is rapid in the presence of oxygen at pH $ 4,
+              | Mn(II) is kinetically stabilized toward oxidation at circumneutral pH, and oxidation by
+              | molecular oxygen requires mineral surface or bacterial catalysis (56). Phylogenetically
+              | diverse Mn(II)-oxidizing microorganisms, including bacteria and fungi, have been
+              | cultured from a wide-range of environments (57). Although this metabolic capability
+              | appears to be widespread, its function is currently unknown. Direct bacterial Mn(II)
+              | oxidation is catalyzed enzymatically, while indirect biological oxidation is driven by non-
+              | enzymatic processes (for example, the oxidation of Mn(II) by metabolic by-products). A
+              | large body of information available regarding biological Mn(II) oxidation is based on
+              | studies involving a low G+C gram positive bacterium, Bacillus sp. SG-1. In genetic
+              | studies of Bacillus sp. SG-1 and other model Mn(II)-oxidizing organisms, it was found
+              | that genes involved in Mn(II) oxidation share high sequence similarity multicopper
+              | oxidases. MnxG, the multi-copper oxidase responsible for Mn(II)-oxidizing activity of
+              | Bacillus sp. SG-1, was localized to the exosporium of after sporulation. Oxidation of
+              | Mn(II) often encrusts the organisms within an Mn oxide shell and is hypothesized to act
+blank         | 
+meta          |                                                                                          7
+text          | as a protective coat against UV radiation, toxic heavy metals, reactive oxygen species,
+              | and viruses (58).
+              |          Synthetic and bacterially produced Mn oxides have been used as effective
+              | oxidants and sorbents in water treatment (59). Manganese oxides are strong oxidants of
+              | trace metals including As (52, 53, 55, 60), Co(II) (61), Cr(III) (62), and organic
+              | molecules.    Many studies examining As cycling and sorption processes in soils have
+              | focused primarily on interactions with Fe(III) oxides because of their greater abundance
+              | (63) and high affinity for As; however, Fe(III) oxides cannot readily oxidize As(III) (53).
+              | Removal of Mn oxides from lake sediments using hydroxylamine hydrochloride or
+              | sodium acetate has been shown to decrease As(III) oxidation appreciably (53, 54, 64)
+              | demonstrating their role as a primary oxidant in a range of natural systems.
+              |          Chemical mechanisms of As(III) oxidation by Mn oxides has been examined
+              | primarily on birnessite, an easily synthesized analog of biotic Mn oxides (57, 65). The
+              | oxidation of As(III) by birnessite proceeds by a two one-electron transfers where Mn(IV)
+              | is reduced to Mn(III) as shown by equation [1]:
+blank         | 
+              | 
+text          |                2MnO2 + H3AsO3 + 2H+ = Mn2+ + H3AsO4 + H2O                  [1]
+blank         | 
+              | 
+text          | followed by reduction of Mn(III) to Mn(IV), producing two equivalents of H3AsO4,
+              | shown in equation [2]:
+blank         | 
+              | 
+text          |                2MnOOH + H3AsO3 + 4H+ = 2Mn2+ + H3AsO4 + 3H2O               [2]
+blank         | 
+              | 
+text          | Arsenic(V) then adsorbs onto the birnessite surface forming a bidentate-binuclear
+              | complex (52, 66). Interestingly, the reductive dissolution of the birnessite surface during
+              | oxidation of As(III) leads to an increase in As(V) sorption capacity due to surface
+              | alterations (52). Although these studies elucidate the mechanisms of As(III) oxidation
+              | and sorption by Mn oxides within batch systems, biologically mediated transformation
+              | of As at redox interfaces is not well understood, where the source of As(III) is
+              | biologically reduced As(V) and may, therefore, be accompanied by other products of the
+meta          |                                                                                          8
+text          | As(V) respiration process.    The effects of these byproducts may have an effect on
+              | oxidation and sorption of As(III) in the presence of oxidants like Mn oxides.
+blank         | 
+              | 
+title         | 1.7 Arsenic sorption dynamics in soil environments
+text          |          Within soils and sediments, various sorbents of As coexist, leading to
+              | competitive adsorption reactions. Application of extended X-ray fine structure (EXAFS)
+              | spectroscopy revealed that As(V) forms bidentate-binuclear complexes on both goethite
+              | and birnessite (22, 52), providing evidence that the two oxides may have competing roles
+              | for As retention in soils. Although numerous past studies have characterized the effects
+              | of As adsorption onto Fe and Mn oxides individually (33-35, 52, 67, 68), whether
+              | preferential adsorption of As onto one oxide over another will occur remains unclear.
+              |          Aqueous As concentrations are often correlated with Fe(II) rather than Mn(II)
+              | (69-71), suggesting that Fe oxides are the primary adsorbent of As. However, there is
+              | evidence that As uptake by soils and sediments is enhanced in the presence of Mn oxides
+              | (72-74). Also, removal of Mn oxides within an Fe-Mn binary-oxide adsorbent developed
+              | for remediation of As lead to appreciably lower As(III) uptake by the adsorbent (75).
+              | Sun et al. (76) examined As adsorption in soils containing ferromanganese nodules/Fe-
+              | Mn oxide mixtures reporting As(III) was oxidized and then adsorbed onto the Fe-Mn
+              | mixed oxide; however, it was not possible to quantify and compare the amount of As
+              | adsorbed on the individual oxide phases. Futhermore, the extent and magnitude of
+              | adsorption can vary depending on oxide type, and the variation in surface coverage will
+              | lead to varying desorption rates.     Tufano and Fendorf (77) demonstrated that the
+              | magnitude of As desorbed from iron (hydr)oxide coated sands varied over time and initial
+              | As loading, providing evidence for the existence of multiple adsorption sites of varying
+              | strengths.
+blank         | 
+              | 
+title         | 1.8 Physical heterogeneity of soils and sediments
+text          |          Physical heterogeneity influences the extent and spatial distribution of oxidative
+              | and reductive processes within soils and sediments, and, as such, may influence the fate
+meta          |                                                                                          9
+text          | and transport of As. Soils are composed of microaggregates fused together by labile
+              | organic matter into macroaggregates (78, 79), which form a complex matrix of transport
+              | mechanisms comprised of advective flow channels between aggregates combined with
+              | diffusion-controlled intra-aggregate transport (80-82).    The rate of intra-aggregate
+              | transport of chemical species such as oxygen from the aggregate exterior decreases
+              | toward the aggregate center due to diminishing pore size, increased tortuosity, and
+              | discontinuities (82).
+              |             Oxygen is further limited within aggregates through microbial respiration,
+              | becoming depleted within millimeters of the aggregate exterior (35, 83). Depletion of
+              | oxygen initiates microbial anaerobic respiration alternative terminal electron acceptors,
+              | including As(V) and Fe(III) and Mn(IV) oxides common to soil aggregates (84). In
+              | many environments, including seasonally saturated soils (85), bioturbated sediments (86),
+              | and forest soils (87), temporary O2 depletion within soil aggregates results from redox
+              | fluctuations (83, 88), where rapid switches in dominant metabolic processes may occur
+              | (86).
+              |             The overall rate of reaction within a wide range of physical system depends
+              | upon the competition between chemical reactivity and diffusive transport of reactants.
+              | Reaction-diffusion processes have been examined for many decades in a wide-range of
+              | disciplines from chemical engineering (89, 90) to medical sciences (91), where accurate
+              | depiction of overall reaction rate requires a comparison of chemical kinetics and mass-
+              | transfer limitations. Similarly, the rate of chemical reactions within soils is greatly
+              | influenced by the availability of reactants within a physically heterogeneous matrix
+              | containing diffusion-controlled transport of species into soil aggregates. Thus, accurate
+              | characterization and quantification of overall chemical transformations within soils
+              | requires simultaneous consideration of chemical reaction kinetics and rates of mass
+              | transfer.
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                       10
+title         | 1.9 Motivation of Research
+text          |          Although past studies have examined and illustrated the role of reduction and
+              | oxidation processes individually, there is currently a lack of information on competitive
+              | redox or coupled processes that may occur within soils. Similarly, the retention of As in
+              | soil matrices is affected by competitive adsorption reactions onto multiple sorption
+              | surfaces. Furthermore, the combined effects of these chemical processes in soils is
+              | complicated by the complex physical structure within which these reactions take place.
+              | In addition, the microbial community responsible for As(V) reduction, a driving factor of
+              | As release in soil environments, has only been briefly explored in disparate locations, and
+              | is currently unknown in many parts of south and southeast Asia. Accordingly, this
+              | dissertation aims to decipher the competitive reactions controlling As transformation and
+              | partitioning within the physically and chemically heterogeneous structure of soils.
+blank         | 
+              | 
+              | 
+              | 
+title         | 1.10 Scope of Research
+text          |          In Chapter 2, I investigate the competitive redox processes controlling As
+              | oxidation and reduction at the interface of Mn oxides and dissimilatory As(V)-reducing
+              | bacteria (DARB); in Chapter 3 I then examine the competitive retention and oxidation of
+              | As(III) on Mn and Fe oxides under anoxic conditions. In Chapter 4, I examine the
+              | combined As redox and sorption processes explored in Chapter 2 and 3 in a single,
+              | physically heterogeneous context of a soil aggregate under various aeration treatments.
+              | Finally, in Chapter 5, I survey the diversity and community structure of dissimilatory
+              | As(V)-reducing bacteria in near-surface sediments of the Mekong Delta in Cambodia,
+              | where porewater and groundwater concentrations of As can be orders of magnitude
+              | greater than the suggested limit for drinking water. Furthermore, I determined whether
+              | porewater concentrations of chemical constituents, including As, Fe, and Mn, directly
+              | impact the community structure of As(V)-reducing bacteria.
+blank         | 
+              | 
+              | 
+meta          |                                                                                         11
+title         | Chapter 2: Competitive microbially and Mn oxide mediated redox processes controlling
+              | arsenic speciation and partitioning
+blank         | 
+              | 
+text          | Within soils and sediments, redox gradients resulting from mass transfer limitations lead
+              | to competitive reduction-oxidation reactions that drive the fate of As. Accordingly, the
+              | objective of this study was to determine the redox cycling of As at the interface of
+              | birnessite (a strong oxidant in soil with a nominal formula of MnOx, where x % 2) and
+              | dissimilatory As(V)-reducing bacteria (strong reductant). Here, we investigate As
+              | reduction-oxidation dynamics in a diffusively controlled system using a Donnan reactor
+              | where birnessite and Shewanella sp. ANA-3 are isolated by a semi- permeable membrane
+              | through which As migrates. Arsenic(III) injected into the reaction cell containing
+              | birnessite is rapidly oxidized to As(V). Arsenic(V) diffusing into the Shewanella chamber
+              | is then reduced to As(III), which subsequently diffuses back to the birnessite chamber,
+              | undergoing oxidation, and establishing a continuous cycling of As. However, we observe
+              | a rapid decline in the rate of As(III) oxidation owing to passivation of the birnessite
+              | surface. Modeling and experimental results show that high [Mn(II)] combined with
+              | increasing [CO32-] from microbial respiration leads to the precipitation of rhodochrosite,
+              | which eventually passivates the Mn oxide surface, inhibiting further As(III) oxidation.
+              | Our results show that despite the initial capacity of birnessite to rapidly oxidize As(III),
+              | the synergistic effect of intense As(V) reduction by microorganisms and the buildup of
+              | reactive metabolites capable of passivating reactive mineral surfaces—here, birnessite—
+              | will produce (bio)geochemical conditions outside of those based on thermodynamic
+              | predictions.
+blank         | 
+              | 
+text          | This chapter has been published in Environmental Science and Technology:
+ref           | Ying, S. C., Kocar, B. D., Griffis, S. D., & Fendorf, S. (2011). Competitive Microbially
+              | and Mn Oxide Mediated Redox Processes Controlling Arsenic Speciation and
+              | Partitioning. Environmental Science & Technology, 45(13), 5572–5579. doi:10.1021/
+              | es200351m
+blank         | 
+              | 
+meta          |                                                                                          12
+title         | Chapter 3: Competitive adsorption of arsenic between goethite and birnessite
+blank         | 
+              | 
+text          | Manganese (Mn) and iron (Fe) oxides are ubiquitous solids in terrestrial systems that
+              | have high sorptive capacities for many trace metals, including arsenic (As). Although
+              | numerous studies have characterized the effects of As adsorption onto Fe and Mn oxides
+              | individually, the fate of arsenic within mixed systems representative of natural
+              | environments is unresolved.      Here, we examine As(III) oxidation and competitive
+              | retention of As on goethite and birnessite using a Donnan reactor, where each oxide is
+              | isolated by a semi-permeable membrane through which arsenic can migrate. To initiate
+              | the Donnan reactor experiments, As(III) is simultaneously added to both chambers.
+              | Arsenic(III) injected into the birnessite chamber is rapidly oxidized to As(V) and then
+              | slowly redistributes across both chambers, while that added to the goethite chamber
+              | undergoes rapid adsorption; As(III) on goethite undergoes desorption and diffusion into
+              | the birnessite chamber and subsequent oxidation to As(V). With increased reaction time,
+              | As(V) is generated and preferentially partitioned onto goethite due to higher sorption
+              | affinity compared to birnessite. Furthermore, reactive transport modeling demonstrates
+              | that the amount of aqueous As available is controlled by the sorption capacity of the
+              | goethite surface, which when saturated, leads to increased aqueous As concentrations.
+              | Our findings show that Mn oxides in soils act as a temporary sorbent of As, but operate
+              | primarily as strong oxidants responsible for transformation of As(III) to As(V), which
+              | can then strongly adsorb on, and is ultimately immobilized by, the surrounding Fe oxide
+              | matrix.
+blank         | 
+              | 
+text          | This chapter has been submitted to Geochimica et Cosmochimica Acta.
+blank         | 
+              | 
+title         | Chapter 4: Arsenic dynamics resulting from distributed microbially- and chemically-
+              | mediated redox processes within Mn-/Fe-oxide constructed aggregates
+blank         | 
+              | 
+text          | The aggregate-based structure of soils imparts physical heterogeneity that that gives rise
+              | to variation in microbial and chemical processes that may influence the speciation and
+meta          |                                                                                        13
+text          | retention of trace elements such as As. To examine the impact of distributed redox
+              | conditions on the fate of As in soils systems, we imposed various redox treatments upon
+              | constructed soil aggregates composed of ferrihydrite- and birnessite-coated sands
+              | presorbed with As(V) and inoculation with the dissimilatory metal reducing bacterium
+              | Shewanella sp. ANA-3. Aeration of the advecting solution surrounding the aggregates
+              | was varied to simulate environmental conditions. We find that diffusion-limited transport
+              | allows reducing conditions to persist in the interior of the aggregate when aerated
+              | treatments are imposed, causing As, Mn, and Fe to migrate from the reduced aggregate
+              | interiors and become immobilized at the aerated exterior region. Upon transition to
+              | anoxic conditions, pulses of As, Mn and Fe are released into the advecting solution
+              | outside of the aggregate in order of energetic yield coupled with lactate oxidation during
+              | microbial respiration. Inversely, release of reduced species from the aggregate into the
+              | advecting solution is inhibited upon transition from aerated to anoxic conditions, where
+              | the oxidized exterior acts as an oxidizing adsorbent barrier. Importantly, we find that
+              | As(III) oxidation by birnessite is appreciable only in the presence of O2, where reductive
+              | dissolution of Mn oxides inhibits oxidation under anaerobic conditions. Our results
+              | demonstrate the importance of considering redox conditions and the physical complexity
+              | of soils in determining the As dynamics, where redox transitions can either enhance or
+              | inhibit As release due to speciation shifts in both sorbents (solubilization versus
+              | precipitation of Fe and Mn oxides) and sorbates.
+blank         | 
+              | 
+text          | This chapter has been submitted to Journal of Environmental Quality.
+blank         | 
+              | 
+title         | Chapter 5: Molecular analysis of arsenate-reducing bacteria within Cambodian
+              | sediments
+blank         | 
+              | 
+text          | Arsenic (As) cycling within soils and sediments of the Mekong Delta of Cambodia is
+              | strongly affected by drastic redox fluctuations caused by seasonal monsoons. Repeated
+              | oxidation and reduction of soils, which contain 10-40 mg kg-1 As, lead to the eventual
+              | downward movement of As to the underlying aquifer.            Extensive flooding during
+meta          |                                                                                        14
+text          | monsoon seasons creates anoxic soil conditions that favor anaerobic microbial metabolic
+              | processes, including arsenate (As(V)) respiration–a process contributing to the
+              | mobilization of As.   Amplification of a highly conserved functional gene encoding
+              | dissimilatory As(V) reductase, arrA, can be used as a molecular marker to detect the
+              | genetic potential for As(V) respiration in environmental samples. However, few studies
+              | have successfully amplified arrA without prior enrichment, and factors influencing
+              | sequence diversity are currently unknown.       In the present study, we examine the
+              | distribution and diversity of arrA genes amplified from multiple study sites within the
+              | Mekong Delta at various soil depths (10, 50, 100, 200, and 400 cm). Additionally, results
+              | from geochemical analysis of soil porewater collected using lysimeters at the
+              | corresponding depths were applied in multivariate analysis to determine whether
+              | variations in arrA sequence could be explained by environmental factors. Here we report
+              | successful amplification, cloning, and sequencing of 223 novel arrA gene sequences from
+              | Cambodia soils without prior enrichment/stimulation, collectively forming a clade that is
+              | phylogenetically distinct from existing sequences in the database.       Application of
+              | permutational MANOVA demonstrates that As and depth variables are most strongly
+              | associated with variations in arrA sequences, whereas a number of other porewater
+              | constituents (including Fe) appeared insignificant.      Our findings demonstrate the
+              | potential for using biogeochemically and ecologically relevant functional genes to
+              | understand operative geochemical processes and, in particular, conditions promoting the
+              | mobilization of arsenic within flooded, tropical soils—systems where arsenic is having a
+              | devastating impact on human health by contaminating regional aquifers.
+blank         | 
+              | 
+title         | 1.11 References
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+meta          |                                                                                       15
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+              | 
+meta          |                                                                                        17
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+              | 
+meta          |                                                                                      18
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+meta          |                                                                                        19
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+              |       in terms of pH and oxidation-reduction potential. Journal Of Geology 68(3):243-
+              |       284 .
+              | 85.   Flessa H & Fischer W (1992) Plant-Induced Changes in the Redox Potentials of
+              |       Rice Rhizospheres. Plant Soil 143(1):55-60 .
+blank         | 
+meta          |                                                                                      20
+ref           | 86.   ALLER R (1994) Bioturbation and Remineralization of Sedimentary Organic-
+              |       Matter - Effects of Redox Oscillation. Chem Geol 114(3-4):331-345 .
+              | 87.   Silver W, Lugo A, & Keller M (1999) Soil oxygen availability and
+              |       biogeochemistry along rainfall and topographic gradients in upland wet tropical
+              |       forest soils. Biogeochemistry 44(3):301-328 .
+              | 88.   Von Fischer J (2002) Separating methane production and consumption with a
+              |       field-based isotope pool dilution technique. Global Biogeochem Cycles.
+              | 89.   Thiele E (1939) Relation between catalytic activity and size of particle. Ind Eng
+              |       Chem 31:916-920 .
+              | 90.   Weisz PB (1973) Diffusion and Chemical Transformation. Science
+              |       179(4072):433-440.
+              | 91.   Roughton F (1957) Relative Importance of Diffusion and Chemical Reaction
+              |       Rates in Determining Rate of Exchange of Gases in the Human Lung, With
+              |       Special Reference to True Diffusing Capacity of Pulmonary Membrane and
+              |       Volume of Blood in the Lung Capillaries. J. Appl. Physiol.
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                     21
+              | 22
+title         | Chapter 2: Competitive Microbially and Mn
+              | oxide Mediated Redox Processes
+              | Controlling Arsenic Speciation and
+              | Partitioning
+blank         | 
+title         | 2.1 Abstract
+text          | The speciation and partitioning of arsenic (As) in surface and subsurface environments is
+              | controlled, in part, by redox processes. Within soils and sediments, redox gradients
+              | resulting from mass transfer limitations lead to competitive reduction-oxidation reactions
+              | that drive the fate of As. Accordingly, the objective of this study was to determine the
+              | fate and redox cycling of As at the interface of birnessite (a strong oxidant in soil with a
+              | nominal formula of MnOx, where x % 2) and dissimilatory As(V)-reducing bacteria
+              | (strong reductant).    Here, we investigate As reduction-oxidation dynamics in a
+              | diffusively-controlled system using a Donnan reactor where birnessite and Shewanella
+              | sp. ANA-3, are isolated by a semi-permeable membrane through which As migrates.
+              | Arsenic(III) injected into the reaction cell containing birnessite is rapidly oxidized to
+              | As(V). Arsenic(V) diffusing into the Shewanella chamber is then reduced to As(III),
+              | which subsequently diffuses back to the birnessite chamber, undergoing oxidation, and
+              | establishing a continuous cycling of As. However, we observe a rapid decline in the rate
+              | of As(III) oxidation owing to passivation of the birnessite surface.        Modeling and
+              | experimental results show that high [Mn(II)] combined with increasing [CO32-] from
+              | microbial respiration leads to the precipitation of rhodochrosite, which eventually
+              | passivates the Mn oxide surface, inhibiting further As(III) oxidation. Our results show
+              | that despite the initial capacity of birnessite to rapidly oxidize As(III), the synergistic
+meta          |                                                                                          23
+text          | effect of intense As(V) reduction by microorganisms and the buildup of reactive
+              | metabolites capable of passivating reactive mineral surfaces—here, birnessite—will
+              | produce (bio)geochemical conditions outside of those based on thermodynamic
+              | predictions.
+blank         | 
+              | 
+title         | 2.2 Introduction
+text          |          Arsenic (As) is a toxic metalloid that can cause human health problems such as
+              | cancer, and eventually death, when consumed chronically. The risk of As exposure,
+              | along with its transport, is highly dependent on its oxidation state. Within soils and
+              | sediments, As typically resides in the trivalent state as the arsenite oxyanion (HxAsO3x-3),
+              | which is more common in reducing environments, or the pentavalent state as the arsenate
+              | oxyanion (HxAsO4x-3) which typically dominates under oxidizing conditions. Arsenic(V)
+              | adsorbs on a range of soil/sediment minerals, with a particular affinity for oxides (used
+              | here to collectively refer to hydroxides, oxyhydroxides, and oxides) of iron, aluminum,
+              | and manganese (1, 2). In contrast, As(III) binds preferentially to iron oxides (3) and,
+              | despite having a greater adsorption maxima at neutral to high pH values (1) it is generally
+              | more mobile than As(V) (4).
+              |          Arsenic(V) retained on the solid phase may be desorbed through biological
+              | reduction of the adsorbing material, such as microbial respiration on iron (5) or
+              | manganese oxides (6), or through direct As(V) reduction (5)–the latter resulting from
+              | microbial respiration or metal detoxification (7). Microbes capable of respiring As(V)
+              | are phylogenically diverse, including members of Proteobacteria and Crenarcheota, and
+              | have been cultured from a diverse range of environments (8); they couple the oxidation of
+              | organic compounds (e.g. acetate, lactate, and formate), or inorganic compounds (e.g. H2,
+              | sulfide), with the reduction of As(V) (8). Detoxification of As is also widespread among
+              | many microbial phyla and has been studied in great detail (see (7) and references
+              | therein), where As(V) is reduced to As(III) by the protein ArsC in the cytoplasm and the
+              | reduced product is then expelled out of the cell through an efflux pump composed of
+              | proteins ArsA and ArsB. The oxidation of As(III) to As(V) can occur in soils under
+meta          | 24
+text          | aerobic conditions, decreasing the concentration of dissolved As(III) and increasing the
+              | extent of adsorption. Furthermore, As oxidation by oxygen is kinetically slow with a
+              | reported half-life of one year (9), and no As(III) oxidation was observed even after 37
+              | days of incubation in demineralized water (10). Arsenic(III) oxidation can be catalyzed
+              | enzymatically by both heterotrophic and chemoautotrophic microbes as a detoxification
+              | mechanism (11) and for growth (12), respectively. Additionally, As oxidation can also
+              | proceed chemically through electron transfer to Mn oxides (2, 13, 14). In terrestrial
+              | environments, Mn oxides compose a smaller fraction of total mass than Fe oxides (15),
+              | but have the capacity to oxidize As(III) to the less toxic and less mobile As(V) state (13,
+              | 16), as shown by studies using natural samples containing Mn oxides from lakes (13),
+              | soils (17), and aquifer materials (18).
+              |          Soils and sediments are composed of a complex network of pores, which often
+              | lead to diffusion-limited mass transport into soil aggregates and large variation in redox
+              | conditions resulting from differing microbial respiration processes; anaerobic processes
+              | may occur within microns of aerobic zones within soils and sediments (19-21) (Figure
+              | 2.1A). As a consequence, oxidants of As(III) such as Mn oxides may reside in close
+              | proximity to reductants, produced microbially or chemically, leading to a tight redox
+              | cycle that will control the fate and transport of As.
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                         25
+text          | Figure 2.1 Schematic illustration of diffusion limited transport and formation of redox gradients
+              | in soil aggregates (A) and schematic overview of arsenic cycling between a suboxic zone
+              | (containing birnessite) and an anoxic zone (containing dissimilatory metal reducing bacteria,
+              | Shewanella) as determined by Donnan cell experiment (B).
+blank         | 
+text          |          Although past studies have illustrated the processes involved in As oxidation
+              | and reduction individually, there is currently a lack of information on competitive or
+              | coupled redox processes that may occur within soils.             Furthermore, the impact of
+              | microbial metabolic products on birnessite reactivity has not been well described. Fischer
+              | et al. (22) demonstrated rhodochrosite precipitation can occur during microbial reduction
+              | of birnessite when coupled with the oxidation of formate. Additionally, rhodochrosite
+              | formation has also been speculated as the reason for the poor correlation of As and Mn in
+              | various environments (23, 24), However, the impact of potential metabolic (or reaction)
+meta          | 26
+text          | products such rhodochrosite formation on arsenic cycling remains unresolved,
+              | particularly within complex physical assemblages such as soils and sediments.
+              | Accordingly, the objective of this study was to determine the fate and redox cycling of As
+              | at the interface of Mn oxides and dissimilatory As(V)-reducing bacteria. To meet this
+              | objective, we employed a modified Donnan reactor which allows the oxidative power of
+              | a Mn oxide, birnessite, to be proximal to As(V)-reducing bacteria, Shewanella sp. ANA-
+              | 3. Arsenic(III) was injected into the reactor, where Shewanella sp. ANA-3 and birnessite
+              | were placed in neighboring reaction chambers separated by a permeable membrane. We
+              | then followed the changes in dissolved As and Mn species as a function of reaction time.
+              | Arsenic(III) within the birnessite chamber is rapidly oxidized to As(V) during the initial
+              | phase of the experiment while the rate of reduction within the Shewanella sp. ANA-3 cell
+              | always exceeds the rate of As(V) diffusion, resulting in As(III) being the sole form of As
+              | in the reduction chamber. As the reaction progresses, the oxidative capacity of the
+              | birnessite diminishes as a consequence of MnCO3 heterogeneous precipitation, resulting
+              | in progressively lower concentrations of As(V). The combination of abiotic Mn(II)
+              | production from As(III) oxidation by birnessite coupled with increased carbonate levels
+              | resulting from biological activity leads to MnCO3 precipitation.
+blank         | 
+              | 
+title         | 2.3 Materials and Methods
+blank         | 
+title         | 2.3.1 MnO2 synthesis and characterization.
+text          |          Birnessite (MnO2) was synthesized using the procedure described in (25).
+              | Briefly, 63 g of KMnO4 were dissolved in 1 L of double deionized (DDI) water, which
+              | was then heated to 90˚C and combined with 66 mL concentrated HCl in a separate 4 L
+              | flask while being vigorously stirred. The reaction continued at 90˚C for 10 min, then
+              | cooled for 30 min before filtering through a vacuum filtration system. Oxides captured
+              | by the filter were re-suspended in DDI water and filtered repeatedly to remove entrained
+              | KMnO4. The MnO2 was dried and crushed using a mortar and pestle and then analyzed
+              | by powder x-ray diffraction using Cu K' radiation. External specific surface area of the
+blank         | 
+meta          |                                                                                        27
+text          | birnessite was determined by single point Brunauer-Emmett-Teller (BET) N2 adsorption
+              | to be 54.9 ± 0.7 m2 g-1.
+blank         | 
+title         | 2.3.2 Abiotic As(III)/birnessite incubations.
+text          |          A 0.5 g L-1 suspension of birnessite was made using N2-purged, autoclaved,
+              | basal salt medium (BSM) (composed of 10 mM PIPES, 2.7 mM KCl, 7.9 mM NaCl, and
+              | 0.4 mM CaCl2!2H2O, pH of 7.1), which was then sonicated for 60 minutes. Arsenic(III)
+              | was added to the birnessite suspension to achieve final concentrations of 50, 100, 205,
+              | 400, 480, 600, 800, 1000, 1300, or 1600 µM. The reaction vessels were then shaken in
+              | the dark at 25˚C for 5 days, after which 10 mL of the slurry was removed, filtered
+              | through a 0.2 µm membrane, and acidified. The aqueous phase As concentration was
+              | then measured using inductively coupled plasma-optical emission spectrometry (ICP-
+              | OES). All experiments were conducted in triplicate. The incubations provided As(III)-
+              | reacted birnessite solids to document morphological differences between incubations of
+              | various As(III) concentrations under abiotic conditions. These batch-incubated solids
+              | were then compared with the morphology of reacted birnessite solids in Donnan reactor
+              | after reactor termination. Solids from As(III)/birnessite batch incubations and Donnan
+              | reactor were captured on nitrocellulose filters then air dried and crushed anaerobically in
+              | preparation for powder x-ray diffraction (XRD) spectroscopy.
+blank         | 
+title         | 2.3.3 Shewanella sp. ANA-3 preparation.
+text          |          200 mL of Shewanella sp. ANA-3 were grown aerobically in autoclaved tryptic
+              | soy broth (30 g L-1 DDI water) at 30°C until late log phase from frozen seed culture
+              | (stored in 20% glycerol at -80°C). Cells were harvested by centrifuging liquid cultures
+              | (5000 x g; 15 min; 25°C), re-suspending the pellet in 30 mL of BSM at pH 7.1, and
+              | repeating the wash steps three times.
+blank         | 
+              | 
+              | 
+              | 
+meta          | 28
+text          | Figure 2.2 Donnan reactor dimensions and experimental set up.
+blank         | 
+title         | 2.3.4 Donnan Cell and Experiments.
+text          |          Experiments were conducted at room temperature (25 ± 2 °C) in a glove bag
+              | with a 95% N2:5% H2 atmosphere in a reaction cell with two chambers separated by a
+              | permeable membrane (0.1 mm isopore polycarbonate filter)—a Donnan cell.            The
+              | reaction cell was constructed with HCl-washed PVC (internal diameter of 5 cm) to form
+              | two 380 mL volume chambers; details, including a schematic diagram of the reactor, are
+              | provided in the supporting information (Figure 2.2). A 5 g L-1 birnessite suspension in
+              | N2-purged BSM was sonically dispersed (90 min), diluted to 1.43 g L-1 with BSM, and
+              | then 337.4 mL added to a reaction chamber. To the other chamber, 100 mL of a 1.9 x 109
+              | cells mL-1 Shewanella sp. ANA-3 culture in BSM was added to 237.4 mL BSM. The
+              | reaction chambers were stirred mechanically with a 3-blade impeller at 400 rpm. Fluid
+              | transport through the semi-permeable membrane is maximized in the presence of
+              | advective flow caused by rapid stirring within the reaction chambers. The reaction was
+meta          |                                                                                     29
+text          | initiated by adding 510 µM sodium meta-arsenite (Na2AsO2), and 3 mM sodium lactate
+              | (NaC3H5O3), simultaneously to both chambers (total volume of 350 mL in each
+              | chamber).
+blank         | 
+title         | 2.3.5 Aqueous phase analysis.
+text          |         At each sampling time, 7 mL of well-mixed slurry from each chamber was
+              | removed, maintaining a constant suspension and cell density, and then filtered through a
+              | 0.2-µm membrane. Total dissolved As and Mn concentrations were then measured by
+              | ICP-OES and As(III)/As(V) were determined using the hydride method described in (26)
+              | as modified by Jones et al. (27).    Finally, lactate and acetate concentrations   were
+              | measured on a separate portion of the filtered solution using ion chromatography (4);
+              | carbonate concentrations were determined by stoichiometric difference in lactate and
+              | acetate concentrations. The pH of solutions within reaction chambers remained constant
+              | due to high concentration of PIPES to maintain a well buffered system.
+blank         | 
+title         | 2.3.6 Solid phase analysis.
+text          |         The concentration of total As on/in the Mn oxide at each time point was
+              | determined by HCl digest. A 2 mL sample of the slurry was collected from each reaction
+              | chamber and placed into acid-washed borosilicate tubes. Samples were dried at 70˚C for
+              | three days and resuspended in 2 mL of concentrated HCl. The solution was heated to
+              | 90˚C and mixed occasionally until solids were completely dissolved.             Arsenic
+              | concentrations in the digested samples were determined using ICP-OES.               The
+              | concentration of As in filtered aqueous phases samples were subtracted from the total As
+              | measured in digested samples to determine the solid phases concentration. Solids were
+              | also collected on ashless filter paper by filtering 1 mL of slurry using a vacuum
+              | apparatus. Filters were stored in Petri plates and kept under anaerobic conditions until
+              | analysis by x-ray absorption near-edge structure (XANES) spectroscopy. XANES spectra
+              | were collected at the Stanford Synchrotron Radiation Lightsource (SSRL) on beamline
+blank         | 
+              | 
+              | 
+meta          | 30
+text          | Table 2.0.1 Reaction network used within the reactive transport model to describe arsenic
+              | dynamics.
+blank         | 
+              | 
+              | 
+              | 
+text          | 11-2 using a Si(220) crystal monochromator and a 30-element Ge solid state detector
+              | array. Spectra were collected from 229 eV below to 277 eV above the As K-edge of
+              | 11867 eV. Energy calibration was performed by scanning a Na3AsO4 standard and
+              | setting the inflection point to 11874 eV. Samples were encased in Kapton and frozen
+              | with liquid nitrogen to prevent oxidation during scans. Linear combination XANES
+              | fitting was done using SIXPACK to determine proportion of As(V) and As(III) while
+              | minimizing X2 values (28).        The change in morphology of birnessite particles was
+              | monitored using scanning electron microscopy (SEM) and elemental analysis gained
+              | through associated energy-dispersive x-ray spectroscopy (EDS) using a FEI XL30 Sirion
+              | SEM in the Stanford Nanocharacterization Laboratory.
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                             31
+title         | 2.3.7 Reactive Transport Modeling.
+text          |          Reactive transport modeling was applied to resolve the complex reaction
+              | network and coupling of diffusion controlled mass transport with adsorption/desorption
+              | equilibria that define the time-dependent partitioning of arsenic.    Following model
+              | calibration, input concentrations of carbon source were varied and oxidation inhibition
+              | terms were removed to perform predictive modeling.           Numerical modeling was
+              | performed using MIN3P, a general purpose reactive transport code capable of coupling
+              | advective-diffusive flow, aqueous and heterogeneous (bio)geochemical reactions, and
+              | solid phase transformations. Details of the general model are described by Mayer et al.
+              | (29).
+              |          The biogeochemical reactions taken into consideration in the Donnan system are
+              | summarized in Table 2.1, and are examined within a one-dimensional simulation
+              | framework (20 cm length) consisting of three cells representing birnessite chamber,
+              | Shewanella sp. ANA-3 chamber, and semi-permeable membrane, with a porosity of 1
+              | and diffusion coefficient of 3.0 x 10-7 m2 s-1. The diffusion rate of As across the
+              | membrane was calibrated to experimental data—480 µM sodium meta-arsenite or sodium
+              | arsenate (Na2HAsO4 • 7H2O) was injected into one chamber of the Donnan reactor; As
+              | was subsequently monitored over time within the injection chamber and the diffusion
+              | chamber. Further details of the modeling approach and diffusion coefficient calculations
+              | are available in supporting information.
+blank         | 
+              | 
+title         | 2.4 Results
+text          |          Competitive oxidation-reduction reactions controlling the fate of As were
+              | examined by injecting As(III) into chambers containing either As(V)-reducing bacteria,
+              | Shewanella sp. ANA-3 (SHEW chamber) or the As(III) oxidizing mineral, birnessite
+              | (BIRN chamber), which were separated by a semi-permeable membrane. Within the
+              | BIRN chamber, As(III) was quickly oxidized to As(V), with As(V)aq concentrations
+              | increasing to 207 µM at 0.7 h and reaching a maximum concentration of 310 µM at 16 h
+blank         | 
+meta          | 32
+text          | (Figure 2.3A). Concomitant with As(V) production, As(III) concentrations decreased
+              | rapidly to 100 µM by 0.7 h. In contrast, As(III) dominates the aqueous speciation
+              | throughout the experiment within the Shewanella chamber, with As(V) concentrations
+              | remaining nearly undetectable (Figure 2.3B).      Total aqueous As in both chambers
+              | decreased over time reflecting an increase in As sorption within the BIRN chamber
+              | (Figure 2.3A and 2.3B). Acid digestion of Shewanella sp. ANA-3 slurries showed no
+              | significant adsorption of As onto the bacteria at any time during the experiment (data not
+              | shown). XANES spectroscopic analysis showed that all of the adsorbed As on birnessite
+              | was As(V) at all time points (data not shown). Speciation of As adsorbed onto bacterial
+              | cells could not be determined due to low As concentrations.
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                        33
+text          | Figure 2.3 Aqueous arsenic concentrations as a function of time in (A) birnessite and (B)
+              | Shewanella chambers, and (C) solid-phase As concentrations in birnessite. Total aqueous arsenic
+              | (black dots), As(V)aq (black squares), and As(III)aq (white squares) are shown. Dotted lines
+              | represent model results.
+blank         | 
+              | 
+              | 
+              | 
+meta          | 34
+text          |          The rate of As oxidation and reduction within the BIRN chamber appears to
+              | have 3 dominant temporal phases (Figure 2.3A); all three periods are reasonably
+              | described with first-order kinetics. From 0 to 0.7 h, [As(III)]aq decreases rapidly, with
+              | commensurate increases in [As(V)]aq, yielding a first-order As(III) oxidation rate constant
+              | of 3.9 h-1. The oxidation rate decreases by 65-fold during the second phase of 1 to 8 h of
+              | reaction, with a rate constant of 0.06 h-1, resulting from the onset of birnessite
+              | transformation and an apparent surface passivation. In the final phase (16 to 132 h),
+              | [As(III)]aq concentrations increase (rather than decrease), albeit at a slow rate, giving a
+              | first-order rate constant of -0.02 h-1. During this reaction period, As(V) reduction rate by
+              | Shewanella sp. ANA-3 dominates over the rate of As(III) oxidation by birnessite.
+              | Manganese(II) concentrations did not follow the three temporal phases shown for As, and
+              | instead total aqueous Mn gradually increased over the course of the experiment in both
+              | reaction chambers, reaching a maximum of 389 µM in the BIRN and 322 µM in the
+              | Shewanella chamber (Figure 2.4A).
+              |          Arsenic uptake on birnessite showed a rapid, linear increase over the first 0.7 h
+              | (Figure 2.3C), with a sorption rate of 0.11 mol As kg-1 h-1. The sorption rate decreased
+              | abruptly as the maximum [As]solid is reached, indicating an apparent cessation in As(III)
+              | oxidation by birnessite.   To investigate the source of oxidation inhibition, we compared
+              | birnessite from the Donnan reactor (after 135 h of reaction) with birnessite abiotically
+              | incubated with 1.6 mM As(III), examining both solids with low angle x-ray diffraction
+              | (XRD) spectroscopy (Figure 2.4B).       The birnessite/ANA-3 Donnan samples display
+              | sharp peaks of high intensity consistent with rhodochrosite (MnCO3), whereas the XRD
+              | data for birnessite incubated with 1.6 mM As(III) and un-reacted birnessite are very
+              | similar and show no indication of rhodochrosite (or any other secondary solid).
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                          35
+text          | Figure 2.4 Total aqueous Mn concentrations in Shewanella and birnessite chambers (squares and
+              | white circles, respectively, right axis) and rhodochrosite saturation index (black circles, left axis)
+              | are shown in (A). Low angle x-ray diffraction patterns for birnessite harvested from Donnan
+              | reactor after experiment termination (B top), birnessite incubated with 1.6 mM As(III) (B
+              | middle), and birnessite with no additions (B bottom) are also shown. Major peaks are labeled as
+              | birnessite, B, and rhodochrosite, R.
+blank         | 
+              | 
+              | 
+meta          | 36
+text          |          The formation of rhodochrosite on birnessite after reaction with As(III) has not
+              | been observed in previous studies.      A unique feature of the present experiment is
+              | microbial respiration (on lactate), which results in increasing (bi)carbonate concentrations
+              | that when combined with Mn(II) generation can lead to precipitation of MnCO3. The
+              | saturation index of rhodochrosite calculated across the experimental timeframe (Figure
+              | 2.4A) illustrates that precipitation becomes favorable. Reactive transport modeling was
+              | used to simulate As(III) oxidation and adsorption dynamics within the Donnan reactor in
+              | order to deconvolute the aqueous and solid phase reactions (reactions considered in the
+              | model are presented in Table 2.1).
+              |          The reaction network used to simulate As reactions within the Donnan cell
+              | (described in detail within the supporting information) capture the change in
+              | concentration of both oxidation states (Figure 2.3).    Both the rapid increase in As(V)
+              | resulting from reaction with birnessite and the cessation of oxidation induced by MnCO3
+              | surface precipitation are well described (Figure 2.3A). Further, the model reasonably
+              | describes the decrease in aqueous As due to adsorption of As(V) on birnessite (Figure
+              | 2.3C) and the production of As(III) by Shewanella (Figure 2.3B). The rate limiting
+              | process throughout the reaction sequence is mass transfer between the reaction cell
+              | induced by diffusion across the semi-permeable membrane—also well represented within
+              | the simulations.
+blank         | 
+              | 
+title         | 2.5 Discussion
+blank         | 
+title         | 2.5.1 Rate Controlling Processes.
+text          |          The fate of arsenic within the Donnan cell is controlled by a combination of
+              | chemical and microbial reactions coupled with diffusion limited transport between the
+              | reaction chambers—controlling processes similar to those operating within soils and
+              | sediments. Previous studies have shown that reaction product formation and distribution
+              | in soil environments can be strongly influenced by diffusion rates (see, for example, (s19,
+              | 20, 21, 29) and summary by (30)). To examine the contributions of diffusion versus
+blank         | 
+meta          |                                                                                          37
+text          | chemical reaction controls on operative transformation rates, we turn to the Thiele
+              | modulus, which has been used within diffusive domains of soil aggregates for such
+              | purposes (see (20) for example). For first-order reactions and constant diffusivity, the
+              | Thiele modulus can be calculated with the following equation:
+blank         | 
+              | 
+              | 
+              | 
+text          | where ! is the dimensionless Thiele modulus, R is the radius of the aggregate (cm), k is
+              | the rate constant (s-1), and De is the effective diffusivity of the reactants within the
+              | aggregate (cm2 s-1). Taking into account aggregate size and effective first-order rate
+              | constants, Tokunaga et al. (20) determined that reactions involving k values greater than
+              | approximately 10-4 s-1 with a diffusional distance >5 mm are diffusion controlled (! > 3)
+              | rather than kinetically limited (! < 0.3).     This is consistent with our findings that
+              | reactions within the reactor are diffusion limited, where applying a diffusional distance of
+              | 10 cm (i.e. half length of reactor) and k values for As(III) oxidation by birnessite and
+              | As(V) reduction by Shewanella sp. ANA-3 as 2.0 x 10-4 s-1 (1) and 1.0 x 10-8 s-1 (27),
+              | respectively, yielded !BIRN = 258 and !SHEW = 1.83. De was calculated from measuring
+              | the diffusion of As(III) and As(V) through the membrane over time.             Because the
+              | diffusional distance is significantly greater than 5 mm, inclusion of the low k for As(V)
+              | reduction rate in the Thiele formula still yields a high ! value. These results confirm that
+              | reactions within the Donnan are representative of diffusively controlled environments.
+blank         | 
+title         | 2.5.2 Competing Redox Processes and Operative Reaction Network.
+text          | Our results illustrate a dynamic competition between bacterial reduction and birnessite
+              | oxidation of arsenic as summarized in Figure 2.1B. Arsenic(III) injected into the Donnan
+              | reactor is quickly oxidized to As(V) by birnessite, which subsequently can diffuse into
+              | the Shewanella chamber and undergo reduction to As(III). The As(III) produced from
+              | bacterial reduction can then diffuse back into the BIRN chamber, where it is re-oxidized,
+              | closing the redox loop between the reducing and oxidizing chambers. Using oxidation
+meta          | 38
+text          | and reduction rates, coupled with adsorption parameters for arsenic on birnessite, along
+              | with diffusion parameters for the reaction cell, we are able to capture (i.e., simulate) the
+              | experimental data in the early time periods of the reaction (Figure 2.3). However, we
+              | observe inhibition of the oxidation step of the cycle after an initial rapid production of
+blank         | 
+              | 
+              | 
+              | 
+text          | Figure 2.5 Simulated aqueous As concentrations in Shewanella and birnessite chambers when
+              | MnCO3 inhibition term is removed in the presence of 3 mM lactate (top) and when lactate
+              | concentrations are decreased to 0.03 mM (bottom) in reactive transport simulations. Total As
+              | (black and white dots), As(V) (black squares), and As(III) (white squares) concentrations are
+              | shown.
+blank         | 
+text          | As(V), and we are unable to adequately describe the experimental trends without
+              | expanding the reaction network.
+              |          Reductive dissolution of birnessite from the oxidation of As(III) leads to solid
+              | phase transformations that can decrease the surface reactivity (2, 14, 31). Generally,
+              | As(III) is first adsorbed onto the oxide surface, followed by the reduction of two Mn(IV)
+              | equivalents to form two Mn(III) intermediates that can be quickly reduced by a second
+blank         | 
+meta          |                                                                                           39
+text          | equivalent of As(III), finally producing the reaction products Mn(II) and As(V), which
+              | can be released into the aqueous phase. Subsequently, Mn(II) produced through the
+              | oxidation of As(III) can re-adsorb onto the birnessite surface, which may progressively
+              | decrease oxidation rates of As(III) (32-34). High concentration of reaction products,
+              | Mn(II) and As(V), have been postulated to result in the formation of manganese(II)-
+              | arsenate precipitates (14, 26, 35). However, manganese arsenate precipitates are unlikely
+              | to be a major product in our experiments given the unperturbed increase in soluble Mn
+              | concentrations within both chambers (Figure 2.4A) and the absence of an As signal in
+              | energy dispersive spectra of the birnessite (data not shown). Instead, XRD data and
+              | saturation indices are consistent with rhodochrosite formation on birnessite, which
+              | subsequently limits its reactivity and leads to a decreased rate of As(III) oxidation (Figure
+              | 2.1B).
+              |          The formation of rhodochrosite is an unexpected product resulting from the
+              | combined redox cycle. Carbonate (inclusive of bicarbonate) is generated from microbial
+              | oxidation of lactate coupled to As(V) reduction while Mn(II) results from As(III)
+              | oxidation by birnessite. As a consequence of these redox products, the saturation index
+              | for rhodochrosite increases rapidly and becomes favorable for precipitation within 10 h of
+              | reaction (Figure 2.4A). The consequence of rhodochrosite formation is an apparent
+              | inhibition of the oxidative capacity of birnessite. By including an inhibition term in the
+              | oxidation rate resulting from MnCO3 formation, the full time-series of the experiment is
+              | well captured by the reactive transport model (Table 2.1, Eqn. 9 and Table A.1, Eqn. 3).
+              |          Interestingly, removal of the rhodochrosite inhibition term from the reaction
+              | network leads to a predicted maintenance of pseudo-steady state conditions within the
+              | two chambers (Figure 2.5).        Arsenic(V) is rapidly formed by birnessite and then
+              | continually reduced to As(III) by Shewanella sp. ANA-3; each reaction chamber reaches
+              | a pseudo-steady state owing to the chemical (oxidation and reduction) rates exceeding
+              | that of diffusion between the cells, and the reactions are perpetuated until either birnessite
+              | or lactate are exhausted. A key factor in the reaction sequence is thus the microbial
+              | generation of (bi)carbonate, along with As(III), which we can further assess through
+blank         | 
+meta          | 40
+text          | reaction simulations.   Diminished carbonate concentrations from decreased lactate
+              | concentration from 3 mM to 1 mM has little impact on the reaction sequence or resulting
+              | As species concentration (Figure 2.6). However, further decreasing lactate to 0.3 mM
+              | and 0.03 mM has the dual-impact of diminishing As(III) production and the extent of
+              | rhodochrosite-induced inhibition of As(V) oxidation by birnessite (Figure 2.6); at 0.03
+              | mM lactate, As(V) becomes the dominant species within both reaction chambers (Figure
+              | 2.5).
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                     41
+text          | Figure 2.6 Simulated As (aq) concentrations in Shewanella (left panel) and birnessite(right
+              | panel) chambers as lactate concentrations were decreased from 1 mM to 0.03 mM in reactive
+              | transport simulations. In the presence of 0.3 mM lactate (middle), the electron donor becomes
+              | limiting at approximately 70 h, as shown by the decreasing As(III) concentrations in the
+              | Shewanella chamber indicating a lowered As(V) reduction rate. At approximately 110 h, the
+              | rate of As(V) reduction decreases below the As(III) oxidation rate within the birnessite chamber.
+              | Although As(III) oxidation by birnessite is inhibited, it is still operative, and therefore As(V)
+              | concentrations begin to increase once again. At 0.03 mM lactate concentrations (bottom), As(V)
+              | reduction by Shewanella cannot be 9 performed and, therefore, carbonate and Mn(II)
+              | concentrations do not reach concentrations great enough to precipitate rhodochrosite, allowing
+              | uninhibited oxidation of injected As(III). Symbols represent total Asaq (dots), As(V)aq (black
+              | squares), As(III)aq (open squares).
+blank         | 
+              | 
+meta          | 42
+text          |         The operative rates of reduction and oxidation within soil micro-sites may shift
+              | depending on aqueous concentrations of reaction products, solid phase transformations
+              | and surface passivation, and microbial activity. Within the Donnan reactor used here to
+              | examine competitive oxidation versus reduction of arsenic, local chemical reaction rates
+              | within each chamber are more rapid than diffusion controlled inter-chamber transfer
+              | rates, leading to As(III) within the bacterial reduction driven chamber and As(V) within
+              | the oxidation driven birnessite cell. However, the combined redox reaction products,
+              | Mn2+ and CO32-, lead to rhodochrosite-induced cessation of oxidation with continued
+              | incubation. Together, the results of this study illustrate the complexity resulting from
+              | redox reaction networks within physically complex soils and sediments that have
+              | diffusionally linked, but operatively separate, biogeochemical environments.
+blank         | 
+              | 
+title         | 2.6 Acknowledgements
+text          |         This research was supported by the Stanford NSF Environmental Molecular
+              | Science Institute (NSF-CHE-0431425) and by the National Science Foundation (grant
+              | number EAR-0952019), and by U.S. EPA STAR graduate fellowship (FP-91678701-3)
+              | to S.C.Y. The authors thank Guangchao Li for analytical assistance and Yoko Masue-
+              | Slowey for laboratory assistance and many helpful discussions. Portions of this research
+              | were carried out at the Stanford Synchrotron Radiation Laboratory, a national user
+              | facility operated by Stanford University on behalf of the U.S. Department of Energy,
+              | Office of Basic Energy Sciences.
+blank         | 
+              | 
+title         | 2.7 References
+blank         | 
+ref           | 1. Dixit, S.; Hering, J. Comparison of arsenic(V) and arsenic(III) sorption onto iron
+              |     oxide minerals: Implications for arsenic mobility. Environ. Sci. Technol. 2003, 37
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+              | 2. Manning, B.; Fendorf, S.; Bostick, B.; Suarez, D. Arsenic(III) oxidation and
+              |     arsenic(V) adsorption reactions on synthetic birnessite. Environ. Sci. Technol.
+              |     2002, 36 (5), 976-981.
+blank         | 
+meta          |                                                                                      43
+ref           | 3.   Gupta, S. K.; Chen, K. Y. Arsenic removal by adsorption. J. Water Poll. Contr.
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+              |      oxidizing bacterium isolated from a gold mine: Phylogenetic, physiological, and
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+              | 13. Oscarson, D.; Huang, P.; Defosse, C.; Herbillon, A. Oxidative power of Mn(IV)
+              |      and Fe(III) oxides with respect to As(III) in terrestrial and aquatic environments.
+              |      Nature 1981, 291 (5810), 50-51.
+              | 14. Scott, M.; Morgan, J. Reactions at oxide surfaces. 1. Oxidation of As(III) by
+              |      synthetic birnessite. Environ. Sci. Technol. 1995, 29 (8), 1898-1905.
+              | 15. Nealson, K.; Saffarini, D. Iron and manganese in anaerobic respiration -
+              |      environmental significance, physiology, and regulation. Annu. Rev. Microbiol.
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+              | 16. Manning, B.; Fendorf, S.; Goldberg, S. Surface structures and stability of
+              |      arsenic(III) on goethite: Spectroscopic evidence for inner-sphere complexes.
+              |      Environ. Sci. Technol. 1998, 32 (5), 2383-2388.
+              | 17. Deschamps, E.; Ciminelli, V.; Weidler, P.; Ramos, A. Arsenic sorption onto soils
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+              |      oxidation of arsenic(III) by aquifer materials. Geochim. Cosmochim. Ac. 2006, 70
+              |      (3), 533-547.
+              | 19. Myrold, D.; Tiedje, J. Diffusional constraints on denitrification in soil. Soil Sci. Soc.
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+              | 20. Tokunaga, T.; Wan, J.; Hazen, T.; Schwartz, E.; Firestone, M.; Sutton, S.;
+              |      Newville, M.; Olson, K.; Lanzirotti, A.; Rao, W. Distribution of chromium
+blank         | 
+meta          | 44
+ref           |       contamination and microbial activity in soil aggregates. J. Environ. Qual. 2003, 32
+              |       (2), 541-549.
+              | 21.   Sexstone, A.; Revsbech, N.; Parkin, T.; Tiedje, J. Direct measurement of oxygen
+              |       profiles and denitrificaiotn rates in soil aggregates. Soil Sci. Soc. Am. J. 1985, 49
+              |       (3), 645-651.
+              | 22.   Fischer, T. B.; Heaney, P. J.; Jang, J. H.; Ross, D. E.; Brantley, S. L.; Post, J. E.;
+              |       Tien, M. Continuous time-resolved X-ray diffraction of the biocatalyzed reduction
+              |       of Mn oxide. Am. Mineral. 2008, 93 (11-12), 1929-1932.
+              | 23.   Ahmed, K. M.; Bhattacharya, P.; Hasan, M. A.; Akhter, S. H.; Alam, S. M. M.;
+              |       Bhuyian, M. A. H.; Imam, M. B.; Khan, A. A.; Sracek, O. Arsenic enrichment in
+              |       groundwater of the alluvial aquifers in Bangladesh: an overview. Appl. Geochem.
+              |       2004, 19, (2), 181-200.
+              | 24.   McArthur, J.; Ravenscroft, P.; Safiulla, S.; Thirlwall, M. Arsenic in groundwater:
+              |       Testing pollution mechanisms for sedimentary aquifers in Bangladesh. Water
+              |       Resour. Res. 2001, 37 (1), 109-117.
+              | 25.   Fendorf, S.; Zasoski, R. Chromium(III) oxidation by delta-MnO2. 1.
+              |       Characterization Environ. Sci. Technol. 1992, 26 (1), 79-85.
+              | 26.   Masscheleyn, P.; Delaune, R.; Patrick, W. Effect of redox potential and pH on
+              |       arsenic speciation and solubility in a contaminated soil. Environ. Sci. Technol.
+              |       1991, 25 (8), 1414-1419.
+              | 27.   Jones, C.; Langner, H.; Anderson, K.; McDermott, T.; Inskeep, W. Rates of
+              |       microbially mediated arsenate reduction and solubilization. Soil Sci. Soc. Am. J.
+              |       2000, 64 (2), 600-608.
+              | 28.   Kocar, B. D.; Herbel, M. J.; Tufano, K. J.; Fendorf, S. Contrasting effects of
+              |       dissimilatory iron(III) and arsenic(V) reduction on arsenic retention and transport.
+              |       Environ. Sci. Technol. 2006, 40 (21), 6715-6721.
+              | 29.   Mayer, K.; Frind, E.; Blowes, D. Multicomponent reactive transport modeling in
+              |       variably saturated porous media using a generalized formulation for kinetically
+              |       controlled reactions. Water Res. Research 2002, 38 (9), 1174.
+              | 30.   Villaverde, J.; Van Beinum, W.; Beulke, S.; Brown, C. D. The kinetics of sorption
+              |       by redtarded diffusion into soil aggregate pores. Environ. Sci. Technol. 2009, 43
+              |       (21), 8227-8232.
+              | 31.   Nesbitt, H.; Canning, G.; Bancroft, G. XPS study of reductive dissolution of 7
+              |       angstrom-birnessite by H3AsO3, with constraints on reaction mechanism. Geochim.
+              |       Cosmochim. Ac. 1998, 62 (12), 2097-2110.
+              | 32.   Zhu, M.; Paul, K. W.; Kubicki, J. D.; Sparks, D. L. Quantum chemical study of
+              |       arsenic(III,V) adsorption on Mn-oxides: Implications for arsenic(III) oxidation.
+              |       Environ. Sci. Technol. 2009, 43 (17), 6655-6661.
+              | 33.   Parikh, S. J.; Lafeerty, B. J.; Meade, T. G.; Sparks, D. L. Evaluating environmental
+              |       influences on As-III oxidation kinetics by a poorly crystalline Mn-oxide. Environ.
+              |       Sci. Technol. 2010, 44 (10), 3772-3778.
+blank         | 
+              | 
+              | 
+meta          |                                                                                          45
+ref           | 34.  Ginder-Vogel, M.; Landrot, G.; Fischel, J. S.; Sparks, D. L. Quantification of rapid
+              |      environmental redox processes with quick-scanning x-ray absorption spectroscopy
+              |      (Q-XAS). Proc. Natl. Acad. Sci. 2009, 106 (38), 16124-16128.
+              | 35. Tournassat, C.; Charlet, L.; Bosbach, D.; Manceau, A. Arsenic(III) oxidation by
+              |      birnessite and precipitation of manganese(II) arsenate. Environ. Sci. Technol.
+              |      2002, 36 (3), 493-500.
+              | 36. Laverman, A.; Blum, J.; Schaefer, K.; Phillips, E.; Lovley, D.; Oremland, R. Growth
+              |      of strain SES-3 with arsenate and other diverse electron-acceptors. Appl Environ
+              |      Microb 1995, 61, (10), 3556-3561.
+              | 37. Morgan, J. J., Chemical equilibria and kinetic properties of manganese in natural
+              |      waters. . Wiley: New York, 1967; p pp. 561-622.
+blank         | 
+              | 
+              | 
+              | 
+meta          | 46
+title         | Chapter 3: Competitive Adsorption of
+              | Arsenic Between Goethite and Birnessite
+blank         | 
+title         | 3.1 Abstract
+text          | Manganese (Mn) and iron (Fe) oxides are ubiquitous solids in terrestrial systems that
+              | have high sorptive capacities for many trace metals, including arsenic (As). Although
+              | numerous studies have characterized the effects of As adsorption onto Fe and Mn oxides
+              | individually, the fate of arsenic within mixed systems representative of natural
+              | environments is unresolved.     Here, we examine As(III) oxidation and competitive
+              | retention of As on goethite and birnessite using a Donnan reactor, where each oxide is
+              | isolated by a semi-permeable membrane through which arsenic can migrate. To initiate
+              | the Donnan reactor experiments, As(III) is simultaneously added to both chambers.
+              | Arsenic(III) injected into the birnessite chamber is rapidly oxidized to As(V) and then
+              | slowly redistributes across both chambers, while that added to the goethite chamber
+              | undergoes rapid adsorption; As(III) on goethite undergoes desorption and diffusion into
+              | the birnessite chamber and subsequent oxidation to As(V). With increased reaction time,
+              | As(V) is generated and preferentially partitioned onto goethite due to higher sorption
+              | affinity compared to birnessite. Furthermore, reactive transport modeling demonstrates
+              | that the amount of aqueous As available is controlled by the sorption capacity of the
+              | goethite surface, which when saturated, leads to increased aqueous As concentrations.
+              | Our findings show that Mn oxides in soils act as a temporary sorbent of As, but operate
+              | primarily as strong oxidants responsible for transformation of As(III) to As(V), which
+              | can then strongly adsorb on, and is ultimately immobilized by, the surrounding Fe oxide
+              | matrix.
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                     47
+title         | 3.2 Introduction
+text          |          Arsenic (As) is a naturally occurring toxic metalloid that is found at hazardous
+              | concentrations in drinking water of many countries within South and Southeast Asia (1-
+              | 3). Long-term consumption of arsenic-containing groundwater within these regions has
+              | lead to chronic poisoning of millions of people who are now manifesting a wide-range of
+              | human health problems including arsenicosis and various types of cancers (4, 5).
+              |          The degree of As mobility in soils and sediments is, in part, governed by the
+              | type of minerals present in the system and the oxidation state of As. Arsenic(III) and
+              | As(V) are the dominant As oxidation states in soils and sediments, where As(V),
+              | typically present as HxAsO4x-3, adsorbs to a wide-range of minerals including iron and
+              | aluminum (hydr)oxides and aluminosilicates minerals; As(III), by contrast, is usually
+              | present as the neutral H3AsO3 species in non-sulfidic environments and preferentially
+              | adsorbs to iron (hydr)oxides (6, 7). Though it has been shown that As(III) can adsorb to
+              | a greater extent on iron oxide surfaces than As(V) (8-10), a substantial portion of As(III)
+              | is bound via weaker complexes (11-13) leading to extensive desorption in the presence of
+              | advective flow (14) Tufano and Fendorf (14) demonstrated that the magnitude of As
+              | desorbed from iron (hydr)oxide coated sands varied over time and initial As loading,
+              | providing further evidence for the existence of multiple adsorption sites of varying
+              | strengths.   These studies show the extent and magnitude of adsorption can vary
+              | depending on oxide type, and the variation in surface coverage will lead to varying
+              | desorption rates.
+              |          Because As(V) and As(III) adsorb to differing extents, redox reactions of As
+              | will have appreciable impacts on adsorption.      Minerals that can oxidize As, such as
+              | manganese (Mn) oxides, will thus alter the extent of As retention. Manganese(III/IV)
+              | oxides are strong oxidants that can oxidize and sequester many trace metals found in
+              | nature (15-17). Arsenic(III) oxidation by Mn oxides leads to reductive alteration of the
+              | surface, leading to enhanced As(V) retention compared to As(V)-treated birnessite (18).
+              | The oxidation reaction of As(III) by birnessite proceeds via two dominant steps where 1)
+blank         | 
+              | 
+              | 
+meta          | 48
+text          | Figure 3.1 Competitive sorption reactions within mass transfer limited soil systems, such as an
+              | rhizosphere aggregates composed of a mixture of Mn oxides along root-zones within an Fe oxide
+              | matrix, is simulated and quantified using the Donnan reactor (bottom panel).
+blank         | 
+              | 
+              | 
+meta          |                                                                                             49
+text          | Mn(IV) is reduced to Mn(III) forming a MnOOH intermediate reaction product followed
+              | by 2) the reaction of As(III) with MnOOH producing Mn2+ and As(V) (19).
+              |          Within soils and sediments, various sorbents of As (such as Fe- and Mn-oxides)
+              | coexist, leading to competitive adsorption reactions. Although numerous past studies
+              | have characterized the effects of As adsorption onto Fe and Mn oxides individually (8,
+              | 14, 18, 20-22), whether preferential adsorption of As onto one oxide over another will
+              | occur remains unclear. Sun et al. (23) examined As adsorption in soils containing
+              | ferromanganese nodules/Fe-Mn oxide mixtures reporting As(III) was oxidized and then
+              | adsorbed onto the Fe-Mn mixed oxide; however, it was not possible to quantify and
+              | compare the amount of As adsorbed on the individual oxide phases.
+              |          The overall rate of reaction within soils and sediments depends upon the relative
+              | rates of chemical reaction as compared to those of mass transfer (24).           For soils and
+              | sediments, structural complexity gives rise to large pores through which solutes flow via
+              | advection intersecting small pores of the bulk matrix in which mass transfer is dominated
+              | by diffusion. Thus, accurate characterization and quantification of overall chemical
+              | transformation within soils require an experimental context that allows for simultaneous
+              | consideration of chemical reaction kinetics and diffusion-limited transport. Herein we
+              | examine reactions controlled by competitive chemical reaction and diffusion processes
+              | using a Donnan reactor composed of reaction cells separated by a semi-permeable
+              | membrane. Diffusive transport controls the exchange of chemical species between the
+              | neighboring cells, while chemical reaction controls the rate within the cells.
+              |          Using the Donnan cell, we examine competitive retention and oxidation of As
+              | on goethite ('-FeOOH) and birnessite under anoxic conditions, where each oxide is
+              | isolated by a semi-permeable membrane through which As can migrate. Using a reactive
+              | transport model (RTM), we are able to determine changes in adsorption dynamics at
+              | environmentally relevant As(III) concentrations and alternate birnessite:goethite ratios.
+              | Our results show that As(III) injected into both chambers is quickly oxidized by
+              | birnessite, with As(V) then diffusing across the semi-permeable membrane where upon it
+              | can adsorb on the goethite surface. Arsenic(III) initially adsorbed on goethite desorbs
+              | and diffuses into the birnessite chamber in response to the concentration gradient
+meta          | 50
+text          | established as a result of As(III) oxidation. Therefore, the speciation of As on goethite
+              | slowly shifts from As(III) to As(V), where the As(III) concentration changes in the
+              | goethite chamber are essentially dependent on the rate of diffusion.       Although the
+              | sorption capacity of birnessite is increased during As(III) oxidation due to surface
+              | alterations, aqueous As(V) concentrations increase when the sorption capacity of goethite
+              | has been exceeded. Our findings show that birnessite acts primarily as an oxidizing agent
+              | to transform the more mobile As(III) to As(V), while goethite acts as the dominant
+              | adsorbent after oxidation.
+blank         | 
+              | 
+title         | 3.3 Material and Methods
+blank         | 
+title         | 3.3.1 Birnessite synthesis and goethite characterization.
+text          |          Birnessite was synthesized by dissolving 63 g of KMnO4 in one liter of doubly
+              | deionized (DDI) water. The solution was heated to 90˚C and combined with 66 mL
+              | concentrated HCl in a separate 4 L flask while being vigorously stirred. The reaction
+              | continued at 90˚C for ten minutes, then cooled for 30 min before filtering through a
+              | vacuum filtration system. Oxides captured by the filter were resuspended in DDI water
+              | and filtered repeated to remove entrained KMnO4. The birnessite was then dried and
+              | crushed using a mortar and pestle.
+              |          Goethite purchased from STREM Chemicals, Inc. (CAS no. 51274-00-1) was
+              | washed with DDI water, dialyzed, then dried and crushed with mortar and pestle. The
+              | identities of both goethite and synthesized birnessite were confirmed by powder X-ray
+              | diffraction analysis using Cu K' radiation. Specific surface area of the birnessite and
+              | goethite were determined by single point Brunauer-Emmett-Teller (BET) N2 adsorption
+              | to be 54.951 ± 0.745 m2 g-1 and 15.526 ± 0.039 m2 g-1, respectively.
+blank         | 
+title         | 3.3.2 Donnan experiment conditions.
+text          |          The experiment was carried out at room temperature under 95% N2:5% H2
+              | atmosphere in an anaerobic glovebag. A Donnan cell was constructed using HCl-washed
+              | PVC pipes (Figure 3.1) with a 0.1-µm polycarbonate filter mounted between the two
+meta          |                                                                                       51
+text          | chambers as the permeable membrane. A concentrated solution of brilliant blue was
+              | added to one side of the cell and allowed to stir for many hours to test for leakages. A
+              | 1:1.35 mass ratio of birnessite:goethite was placed into the Donnan reactor and oxides
+              | were suspended in 350 mL of N2-purged 10 mM PIPES (pH 7.0) and 0.1 M NaCl and
+              | stirred at approximately 400 rpm for 5 h before As(III) was added. The reaction was
+              | initiated by adding 480 and 40 µM sodium meta-arsenite, NaAsO2 (Sigma-Aldrich), with
+              | continued stirring. Two concentrations were used to test the distribution of As between
+              | the two oxide sorbents when total As available in the system is above (480 µM) and
+              | approximately equal (40 µM) to the adsorption maximum of the two oxides (as inferred
+              | from adsorption isotherms).      After As(III) was added, the pH was monitored and
+              | adjusted using 3 M HCl.
+blank         | 
+title         | 3.3.3 Adsorption isotherms.
+text          |          Adsorption isotherms were conducted on birnessite and goethite to estimate
+              | maximum adsorption capacities of As(V) and As(III) at pH 7. A 0.5 g L-1 suspension of
+              | birnessite or goethite was made using N2-purged, autoclaved, basal salt medium (BSM)
+              | (composed of 10 mM PIPES, and 0.1 M NaCl), which was then sonicated for 60 minutes.
+              | Arsenic(III) or As(V) was added to the oxide suspensions to achieve final concentrations
+              | of 10, 20, 50, 100, 205, 400, 480, 600, 800, 1000, 1300, or 1600 µM. The reaction
+              | vessels were then shaken in the dark at 25˚C for 5 d, after which 10 mL of the slurry was
+              | removed, filtered through a 0.2 µm membrane, and acidified. The aqueous phase As
+              | concentration was then measured using inductively coupled plasma-optical emission
+              | spectrometry (ICP-OES). All experiments were conducted in triplicate. Adsorption data
+              | were then fit to the Langmuir isotherm, providing the adsorption maxima. Adsorption
+              | maxima of As(V) on birnessite was 146.2 µmol As g-1 birnessite and 118.3 µmol As g-1
+              | goethite. In As(III)/birnessite incubations, birnessite is reduced during As(III) oxidation,
+              | providing an altered adsorption isotherm where the adsorptive capacity of birnessite
+              | increases with increasing As(III), consistent with previous experiments. For incubations
+              | containing 10 to 600 µM As(III), this increase was quantified as a linear regression of the
+blank         | 
+              | 
+meta          | 52
+text          | amount of As(III) added into the incubation and the amount adsorbed at equilibrium
+              | (Table 1, equation 1).
+blank         | 
+title         | 3.3.4 Aqueous phase analysis.
+text          |          At each sampling time, 5 mL of well mixed slurry from each chamber was
+              | removed and filtered through a 0.2-µm membrane and As, Fe, and Mn concentrations
+              | were measured using inductively coupled plasma optical emission spectrometry (ICP-
+              | OES). Total As concentrations were also measured in samples using hydride generation
+              | inductively coupled plasma spectrometry (HG-ICP-AES) where 3 mL of sample was
+              | acidified (3 M HCl), reacted with 5% (w/v) KI, and mixed with 0.6% (w/v) NaBH4/0.5%
+              | (w/v) NaOH in a reaction coil.      Three mL of filtrate were used for As(III)/As(V)
+              | speciation using the method from Masscheleyn et al. (26) as modified by Jones et al. (27).
+              | While purging with N2, 0.6 mL of 2 M Tris (pH 6.0) was added to 3 mL of sample. After
+              | Tris was thoroughly mixed into sample, two additions of 0.3 mL of 3% (w/v) NaBH4 in 1
+              | M NaOH was added to sample tube, with 5 minutes of N2 purging between additions.
+blank         | 
+title         | 3.3.5 Solid phase analysis.
+text          |          The concentrations of total arsenic adsorbed onto the oxide solid phases were
+              | determined by HCl digestion; 2 mL of slurry was collected from each reaction chamber
+              | and placed in acid-washed borosilicate tubes. Samples were dried at 80˚C for 3 d and re-
+              | suspended in 2 mL of concentrated HCl. The solution was heated to 90˚C and vortexed
+              | occasionally until the oxides were completely dissolved; As concentration was then
+              | measured in the digested samples after ca. 10 h using ICP-OES as described above.
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                        53
+text          | Table 3.1 Reactions and parameters considered in reactive transport modeling using MIN3P.
+blank         | 
+              | 
+              | 
+              | 
+text          | Solids were also collected on ashless membranes by filtering 1 mL of slurry using a
+              | vacuum apparatus. Filters were stored in Petri plates and kept under anaerobic conditions
+              | until analysis by X-ray absorption near-edge structure (XANES) spectroscopy. XANES
+              | spectra were collected at the Stanford Synchrotron Radiation Lightsource (SSRL) on
+              | beamline 11-2 using a Si(220) crystal monochromator and a 30-element Ge solid state
+              | Detector Array. Spectra were collected from 229 eV below to 277 eV above the As K-
+              | edge of 11867 eV. Energy calibration was performed by scanning a sodium arsenate
+              | dibasic hepta-hydrate (Na2HAsO4 • 7H2O) standard and setting the inflection point to
+              | 11874 eV. Samples were encased in Kapton and frozen with liquid nitrogen to prevent
+              | oxidation during scans; no changes were noted between successive scans.                Linear
+meta          | 54
+text          | combination XANES fitting was done using SIXPACK (25) to determine proportion of
+              | As(V) and As(III) while minimizing X2 values (20).
+blank         | 
+title         | 3.3.6 Reactive transport modeling.
+text          | Reactive transport modeling was applied to first constrain the probable rates and
+              | mechanisms of reactions in our system.               Following model calibration, input
+              | concentrations of As were varied and the ratio of birnessite:goethite was decreased to
+              | perform predictive modeling.      Numerical modeling was performed using MIN3P, a
+              | general purpose reactive transport code capable of coupling advective-diffusive flow,
+              | aqueous and heterogeneous (bio)geochemical reactions, and solid phase transformations.
+              | Details of the general model are described by Mayer et al. (36).
+              |          The biogeochemical reactions taken into consideration in the Donnan system are
+              | summarized in Table 1, and are examined within a one-dimensional simulation
+              | framework (20 cm length) consisting of three cells representing birnessite chamber,
+              | goethite chamber, and a semi-permeable membrane with a porosity of 1 and diffusion
+              | coefficient of 8.75 x 10-8 m2 s-1. The diffusion rate of As across the membrane was
+              | calibrated to experimental data—480 µM or 40 µM As(V) or As(III) was injected into
+              | one chamber of the Donnan reactor. Arsenic was subsequently monitored over time in
+              | the injection chamber and the diffusion chamber. Further details of the modeling
+              | approach and diffusion coefficient calculations are available in supporting information.
+blank         | 
+              | 
+title         | 3.4 Results
+blank         | 
+title         | 3.4.1 Aqueous As dynamics within Donnan reactor.
+text          |          Competitive adsorption of As on two metal oxide sorbents, goethite ('-FeOOH)
+              | and birnessite (MnO2), were examined by injecting As(III) into a Donnan reactor, where
+              | the two oxides were placed in chambers separated by a semi-permeable membrane. At
+              | both 480 and 40 µM As(III), As was rapidly oxidized by birnessite to As(V), which then
+              | diffused into the goethite chamber at a flux of 8.75 x 10-8 m2 s-1 (Figure 3.2).
+blank         | 
+              | 
+meta          |                                                                                            55
+text          | Figure 3.2 Aqueous arsenic concentrations in goethite chamber (right panel) and birnessite
+              | chamber (left panel) when 480 µM (top) and 40 µM (bottom) As(III) is injected into the reactor.
+              | Total aqueous arsenic (black dots), As(V)aq (white squares), and As(III)aq (black squares) are
+              | shown. Dotted line represents model results for aqueous As(V) and dashed line for aqueous
+              | As(III).
+blank         | 
+text          | At the higher As(III) concentration (480 µM), As(V)(aq) concentrations within the
+              | birnessite chamber increase from 102 µM to 353 µM within the first 20 min of As(III)
+              | addition; a concomitant rapid decrease in As(III) concentration from 295 µM to 41 µM
+              | occurred within the same time period. Correspondingly, As(V) concentrations in the
+              | goethite chamber slowly increased from 1.55 µM to 4.62 µM within the first hour of
+              | reaction, and As(III) decreased at the same rate. Similarly, when 40 µM As(III) is
+              | injected into the reactor, a rapid spike in As(V) is observed within 20 minutes in the
+              | birnessite chamber with As(III) being undetectable almost immediately after injection;
+blank         | 
+meta          | 56
+text          | while in the goethite chamber, As(V) increases at a rate consistent with that predicted for
+              | diffusion.    The point at which As(III) and As(V) are at approximately equal
+              | concentrations in the goethite chamber occurs 23 h earlier at high As(III) concentrations
+              | (8 h) than at low concentrations (32 h). Sorption of As on solid phases is reflected in the
+              | decrease of As from the aqueous phase where 16% of 480 µM As and 95% of 40 µM As
+              | injected was depleted from the aqueous phase by 135 h.
+blank         | 
+text          | Figure 3.3 Solid phase arsenic concentrations in goethite (white circles) and birnessite chamber
+              | (black circles) when 480 µM (A) and 40 µM (B) As(III) is injected into the reactor. Dotted line
+              | represents model results for sorption onto birnessite and dashed line for sorption onto goethite.
+blank         | 
+              | 
+              | 
+              | 
+title         | 3.4.2 Arsenic sorption onto goethite and birnessite.
+text          |          At the higher As(III) concentration (480 µM As), on a mass basis of the solid, a
+              | higher concentration of As was associated with birnessite than goethite throughout the
+              | experiment–an average concentration of 110 µmol g-1 sorbed on birnessite and 21.6 µmol
+              | g-1As on goethite after (pseudo) steady state is reached at approximately 52 h (Figure
+              | 3.3). Conversely, at the lower As(III) concentration (40 µM As), a greater concentration
+              | of As is initially (from 0 to 2.2 h) associated with birnessite, but then progressively
+              | transfers (desorption, transport, re-adsorption) onto to goethite until a steady-state
+              | concentration of 13.35 µmol g-1 is reached after 52 h of reaction (Figure 3.3).
+blank         | 
+              | 
+              | 
+meta          |                                                                                                     57
+text          |          At both concentrations examined, the proportion of As(III) and As(V) on the
+              | goethite changed during reaction progression—As(III) concentration decreased while,
+              | concomitantly, As(V) increased at a rate-dependent on diffusional transport across the
+              | semi-permeable membrane separating the reaction cells. Therefore, As(III) and As(V)
+              | ligand exchange on the goethite surface is more rapid than the rate of mass transfer that is
+              | controlled by diffusion across the semi-permeable membrane. XANES spectroscopic
+              | analysis of birnessite solids illustrated that all of the As was present in the pentavalent
+              | state (data not shown).
+              |          Oxidation of As(III) by birnessite leads to the reductive dissolution/
+              | transformation of birnessite via Mn(IV) reduction to Mn(II), which can subsequently be
+              | adsorbed by the remaining birnessite or released into the aqueous phase. The Mn(II) that
+              | is released into the aqueous phase can diffuse from the birnessite chamber into the
+              | goethite chamber and absorb on the iron oxide. Although limited within the 40 µM
+              | As(III) system, reaction with 480 µM As(III) resulted in a progressive increase in Mn(II)
+              | concentration on goethite (Figure 3.4B), with the rate of accumulation being, again,
+              | diffusion controlled across the semi-permeable membrane.
+blank         | 
+              | 
+              | 
+              | 
+text          | Figure 3.4 Fraction of As(V) (white symbols) and As(III) (black symbols) adsorbed on goethite
+              | as determined by XANES analysis when 480 µM (circles) or 40 µM As(III) (squares) was added
+              | into Donnan reactor (A). Total solid phase Mn concentrations in goethite digestions when 480
+              | µM (black circles) or 40 µM As(III) (white triangles) is added into Donnan reactor (B).
+blank         | 
+              | 
+              | 
+              | 
+meta          | 58
+title         | 3.4.3 Control experiments and reactive transport model calibration.
+text          |          We use reactive transport modeling to discern the influence of specific reactions
+              | within a multi-process system and to expand the experimental condition through
+              | predictive modeling. In order to calibrate the reactive transport model, a set of diffusion
+              | and adsorption reactions were executed to acquire reaction rates and to constrain the
+              | probable mechanisms in the system. To determine the diffusion rate of As(V) and
+              | As(III), 480 µM As was injected into one chamber of the Donnan reactor and allowed to
+              | diffuse into the neighboring chamber in the absence of Fe or Mn solids (Figure B.1). The
+              | diffusion rate of As(V) and As(III) were nearly identical and thus no differences in
+              | diffusion rates were applied in the RTM.
+blank         | 
+              | 
+              | 
+              | 
+text          | Figure 3.5 Arsenic(III) input concentration in As(III) versus sorbed As on birnessite
+              | As(III)/birnessite incubations
+blank         | 
+              | 
+text          |          To examine the transport and binding of As species on birnessite or goethite,
+              | 480 µM As(III) or As(V) was injected into one chamber and allowed to diffuse into the
+              | second chamber containing either birnessite or goethite (no As was added into the Fe/Mn
+              | oxide chamber directly) (Figure B.2). The concentration of As adsorbed on birnessite
+              | was consistently higher than the amount adsorbed on goethite for both As(III) and As(V)
+              | treatments. Manning et al. (18) showed that reductive dissolution of birnessite during the
+blank         | 
+meta          |                                                                                         59
+text          | oxidation of As(III) lead to rearrangement of the oxide structure, effectively increasing
+              | the sorptive capacity of the Mn solid. Our experiments are consistent with this finding,
+              | where the concentration of As sorbed (adsorbed or incorporated) onto/into birnessite is
+              | greater when it is reacted with As(III) than with As(V) (Figure B.2). The difference in
+              | quantity of adsorbed As can then be used to calculate effective sorption sites (whether
+              | they be truly adsorption sites or sites of As(V) incorporation into the surface structure)
+              | created per mole of As(III) oxidized. In combination with As(III)/birnessite altered
+              | sorption isotherm (Figure 3.5), we determined that 0.16 sorption sites are created for each
+              | mol of As(III) reacted per mol MnO2 (Figure 3.5).
+blank         | 
+              | 
+title         | 3.5 Discussion
+text          |          Iron and manganese oxides are ubiquitous solids found in terrestrial
+              | environments that can act as sorbents for trace metals.         Our results illustrate the
+              | concentration dependence of competitive sorption for As on multiple sorbents along with
+              | the compounding impacts of As(III) oxidation and associated changes to the Mn-oxide
+              | solid. Owing to the rapid oxidation of As(III) by birnessite, the final distribution of As
+              | results from the competing affinities of goethite and reductively modified birnessite
+              | surfaces for As(V); despite having a non-oxidizing sorbent separated by a semi-
+              | permeable membrane, As(III) is fully oxidized to As(V). The resulting As(V) sorbed to a
+              | greater extent on birnessite than goethite in the presence of high As concentration, while,
+              | in contrast, goethite is the dominant adsorbent at lower concentrations (Figure 3.3).
+              | Treatment of Fe-Mn binary oxide sorbent with a strong reductant was shown to increase
+              | As(V) sorption, while decreasing sorption of As(III) (26).         Similarly, our results
+              | demonstrate the alteration of the birnessite surface upon As(III) oxidation promotes
+              | As(V) sorption–consistent with the findings of Manning et al. (2002)—with the increase
+              | in sorption being proportional to the amount of As(III) reacted with the birnessite (Figure
+              | 3.5).
+              |          Although mechanistically unfounded, we are able to describe competitive
+              | sorption between the two oxides with the RTM using two site types, weak and strong, on
+blank         | 
+meta          | 60
+text          | each oxide. The adsorption constants for each oxide and site type are provided in Table
+              | 1. In the presence of low As concentrations, goethite sorbs greater concentration of As
+              | than birnessite; however, at high As concentrations, adsorption sites on goethite surface
+              | become saturated while a higher concentration of As is achieved on birnessite due to
+              | reductive alteration (induced by reaction with As(III) and creation of high-energy sites).
+blank         | 
+              | 
+              | 
+              | 
+text          | Figure 3.6 Predictive modeling results of aqueous As with 0.4, 4, 10, and 20 µM As input in the
+              | birnessite chamber (A) and goethite chamber (B) with insets showing expanded regions. As(III)
+              | (dashed lines), As(V) (solid lines) are shown. (C) Predictive modeling results of As adsorbed onto
+              | birnessite with 20, 10, 4, and 0.4 µM of As(III) input. (D) Predictive modeling results of As
+              | adsorbed onto goethite with 20, 10, 4, and 0.4 µM of As(III) input.
+blank         | 
+text          |          Using the diffusion and oxidation rates and constants derived from experimental
+              | controls, we were able to simulate the experimental data reasonably well throughout the
+              | entire reaction period (Figure 3.2). Reactions considered in the RTM are presented in
+              | Table 1 and the parameterization shown in the EA. Model simulations confirmed that the
+              | shift in aqueous As speciation in the goethite chamber is determined solely by the
+blank         | 
+meta          |                                                                                                61
+text          | diffusion rate at both concentrations tested due to the rapid rate of As(III) oxidation by
+              | birnessite and rapid adsorption/desorption, relative to the rate of mass transfer, of both As
+              | species (Figure 3.2). Our findings are in agreement with past findings that As(III)
+              | oxidation is exclusively controlled by Mn oxides under anoxic conditions and proceeds
+              | rapidly (27), greatly exceeding the rate of diffusive transport. Arsenic sorption on both
+              | birnessite and goethite were also well described at both concentrations using sorption
+              | constants listed in Table 1. (Figure 3.3).
+              |          Using RTM simulations we are able to provide an expansive prediction of
+              | arsenic behavior for variations in arsenic concentration or mass ratios of competitive
+              | sorbents. For As concentrations ranging from 20 to 0.4 µM, As(III) is rapidly oxidized
+              | by birnessite, with the rate of oxidation increasing with As concentration in agreement
+              | with the findings of Oscarson et al. (27). Depletion of As(III) within the birnessite
+              | chamber establishes a concentration gradient that leads to As(III) migration from the
+              | goethite chamber into the birnessite chamber where it is rapidly oxidized. By contrast,
+              | within the concentrations explored here (20 to 0.4 uM), As(V) retention is dominated by
+              | goethite, with As(V) generated in the birnessite chamber diffusing into the goethite
+              | chamber and adsorbed (Figure 3.6); sorption is dominated by goethite across this
+              | concentration range. Arsenic(III) sorption by Fe oxides is enhanced in the presence of
+              | Mn oxides predominantly due to transformation to As(V), while also benefiting from the
+              | creation of sorption sites (26, 28)
+              |          Aqueous concentrations of As appear to be controlled by the combined
+              | oxidation of As(III) by birnessite and subsequent adsorption of As(V) on goethite.
+              | Although As(V) is retained on/in birnessite at the highest As concentration explored (480
+              | mM), regulation of dissolved concentrations are limited (Figure 3.2). To further test the
+              | premise that goethite dominates the retention of As, but is dependent on As(III) oxidation
+              | by birnessite, we conducted a series of RTM simulations at varying birnessite to goethite
+              | mass ratios with initial As(III) concentrations at 40 mM. When the mass of birnessite is
+              | decreased relative to goethite, greater aqueous concentrations of As result (Figure 3.7).
+              | Aqueous As(III) concentrations are negligible at all ratios, but as birnessite decreases the
+              | rate of oxidation concomitantly decreases and results in proportionally higher total
+meta          | 62
+text          | dissolved As concentrations. Thus, with decreasing Mn oxide concentrations (relative to
+              | Fe oxides), As(III) oxidation is restricted and a resulting increase in aqueous arsenic is
+              | expected.
+blank         | 
+              | 
+              | 
+              | 
+text          | Figure 3.7 Predictive model simulations of As concentrations in the aqueous phase and solid
+              | phase in birnessite chamber (A and C respectively), and aqueous and solid phase in goethite
+              | chamber (B and D respectively) at 1:10, 1:50, 1:100 goethite to birnessite ratios in the presence of
+              | 40 µM As(III). Arsenic(III) is instantly oxidized. Aqueous As(V) (solid lines) and aqueous
+              | As(III) (dashed lines) are shown. Lines have multiple labels if data overlaps.
+blank         | 
+text          | The mass ratio of Mn oxides to Fe oxides can vary extensively, ranging from greater than
+              | 1:1 to less than 1:100, with iron generally being 5 to 10 times more abundant than
+              | manganese (15). Decreasing Mn oxide content decreases the rate of As(V) production,
+              | with subsequent adsorption onto the surrounding Fe oxide matrix. However, even at low
+              | Mn to Fe oxide ratios (1:100), the oxidation rate is significantly greater than the rate of
+              | diffusion limited transport; hence, variation in mass ratio has little effect upon the
+blank         | 
+meta          |                                                                                                  63
+text          | speciation of As adsorbed on Fe oxides. For example, the mass ratio of Mn to Fe oxides
+              | may shift under anaerobic conditions, where reductive dissolution of Mn occurs prior to
+              | Fe oxide reduction as a result of greater thermodynamic favorability; the rate of As(V)
+              | production in the soil matrix as a whole will be nearly unaffected by the decreasing
+              | presence of Mn oxides until complete dissolution of the Mn oxides. The total sorption
+              | capacity of the soil is controlled by the available sorption sites on Fe oxide; therefore,
+              | initial removal of Mn oxides will impact As mobility due to decreased sorption strength
+              | (i.e. As(V) adsorbs more strongly on Fe oxides than As(III)) but has little influence on
+              | sorption extent).
+blank         | 
+              | 
+title         | 3.6 Conclusions
+text          |          Arsenic migration through soil is determined, in part, by the oxidation state of
+              | arsenic, sorption capacity of the matrix, and transport mechanisms. Our experimental
+              | results combined with reactive transport modeling demonstrate that the rate of mass
+              | transfer dominates adsorption/desorption and oxidation, of which both processes are
+              | rapid relative to diffusion limited transport. Further, As(V) production is decreased as
+              | the mass of birnessite available decreases, consistent with previous findings (18, 29).
+              | Scott and Morgan (29) demonstrated As(V) is quickly desorbed after rapid oxidation by
+              | birnessite resulting in high aqueous As concentrations. Due to the relatively rapid rate of
+              | As(III) oxidation by birnessite, strong diffusional gradients are produced leading to
+              | desorption and transport of As(III) that is initially sorbed on goethite. Arsenic(III) that is
+              | removed from the surface of goethite is diffusionally transported and then rapidly
+              | oxidized by Mn oxides. The resulting As(V) then diffuses back toward the goethite and
+              | undergoes adsorption, consistent with previous findings that Mn oxides enhance the
+              | uptake of As by Fe oxides through the conversion of As(III) to As(V) (28, 31, 32).
+              | Although Mn-oxides exhibit an increase retention capacity for As(III) owing to surface
+              | alteration upon oxidation to As(V), the aqueous concentrations of As increase
+              | appreciably when the sorption capacity of goethite is exceeded, indicative of a lower
+              | affinity for Mn oxides, even after reductive modification, for As(V) relative to Fe oxides
+blank         | 
+meta          | 64
+text          | such as goethite. Thus, within natural environments, Mn oxides served to transform
+              | (oxidize) As(III) to the stronger adsorbate, As(V), where upon Fe oxide act as dominant,
+              | high-affinity sinks for As.
+blank         | 
+              | 
+title         | 3.7 Acknowledgements
+text          |          This research was supported by the Stanford NSF Environmental Molecular
+              | Science Institute (NSF-CHE-0431425) and by the National Science Foundation (grant
+              | number EAR-0952019), and by U.S. EPA STAR graduate fellowship (FP-91678701-3)
+              | to S.C.Y. The authors thank Guangchao Li for analytical assistance and Yoko Masue-
+              | Slowey for helpful input and discussions and helping with very late night sampling.
+              | Portions of this research were carried out at the Stanford Synchrotron Radiation
+              | Laboratory, a national user facility operated by Stanford University on behalf of the U.S.
+              | Department of Energy, Office of Basic Energy Sciences.
+blank         | 
+              | 
+title         | 3.8 References
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+blank         | 
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+blank         | 
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+blank         | 
+ref           | 4.    Chen Y, Ahsan H (2004) Cancer burden from arsenic in drinking water in
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+blank         | 
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+blank         | 
+ref           | 6.    Gupta S, Chen K. (1978) Arsenic Removal by Adsorption. J Water Pollut Con F
+              |       50:493–506.
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                        65
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+blank         | 
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+blank         | 
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+blank         | 
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+blank         | 
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+              |       oxidation of manganese. Adv Appl Microbiol 33:279–318.
+blank         | 
+ref           | 16.   Toner B, Manceau A, Webb S, Sposito G (2006) Zinc sorption to biogenic
+              |       hexagonal-birnessite particles within a hydrated bacterial biofilm. Geochim
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+blank         | 
+ref           | 17.   Young L, Harvey H (1992) The relative importance of manganese and iron-oxides
+              |       and organic-matter in the sorption of trace-metals by surficial lake-sediments.
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+blank         | 
+ref           | 18.   Manning B, Fendorf S, Bostick B, Suarez D (2002) Arsenic(III) oxidation and
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+              | 
+meta          | 66
+ref           | 19.   Nesbitt H, Canning G, Bancroft G (1998) XPS study of reductive dissolution of 7
+              |       angstrom-birnessite by H3AsO3, with constraints on reaction mechanism.
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+blank         | 
+ref           | 20.   Kocar BD, Herbel MJ, Tufano KJ, Fendorf S (2006) Contrasting effects of
+              |       dissimilatory iron(III) and arsenic(V) reduction on arsenic retention and transport.
+              |       Environ Sci Technol 40:6715–6721.
+blank         | 
+ref           | 21.   Manning B, Fendorf S, Goldberg S (1998) Surface structures and stability of
+              |       arsenic(III) on goethite: Spectroscopic evidence for inner-sphere complexes.
+              |       Environ Sci Technol 32:2383–2388.
+blank         | 
+ref           | 22.   Masue-Slowey Y, Kocar BD, Jofre SAB, Mayer KU, Fendorf S (2011) Transport
+              |       Implications Resulting from Internal Redistribution of Arsenic and Iron within
+              |       Constructed Soil Aggregates. Environ Sci Technol 45:582–588.
+blank         | 
+ref           | 23.   Sun X, Doner H (1998) Adsorption and Oxidation of Arsenite on Goethite. Soil
+              |       Science 163.
+blank         | 
+ref           | 24.   Aharoni C, Sparks D, Levinson S (1991) Kinetics of soil chemical reactions:
+              |       Relationships between empirical equations and diffusion models. Soil Sci Soc Am
+              |       J.
+blank         | 
+ref           | 25.   Webb SM (2005) SIXpack: a graphical user interface for XAS analysis using
+              |       IFEFFIT. Phys Scripta T115:1011–1014.
+blank         | 
+ref           | 26.   Zhang JJ, Smith KR (2007) Household air pollution from coal and biomass fuels in
+              |       China: Measurements, health impacts, and interventions. Environ Health Persp
+              |       115:848–855.
+blank         | 
+ref           | 27.   Oscarson D, Huang P, Defosse C, Herbillon a (1981) Oxidative Power of Mn(Iv)
+              |       and Fe(Iii) Oxides with Respect to as(Iii) in Terrestrial and Aquatic Environments.
+              |       Nature 291:50–51.
+blank         | 
+ref           | 28.   Deschamps E, Ciminelli V, Weidler P, Ramos A (2003) Arsenic sorption onto
+              |       soils enriched in Mn and Fe minerals. Clay Clay Miner 51:197–204.
+blank         | 
+ref           | 29.   Scott M, Morgan J (1995) Reactions at Oxide Surfaces .1. Oxidation of as(Iii) by
+              |       Synthetic Birnessite. Environ Sci Technol 29:1898–1905.
+blank         | 
+ref           | 30. Nealson, K., Saffarini, D. (1994). Iron and Manganese in Anaerobic Respiration -
+              |      Environmental Significance, Physiology, and Regulation. Annual Review Of
+              |      Microbiology, 48, 311–343.
+blank         | 
+              | 
+              | 
+meta          |                                                                                        67
+ref           | 31. Deschamps, E., Ciminelli, V., & Holl, W. (2005). Removal of As(III) and As(V)
+              |      from water using a natural Fe and Mn enriched sample. Water Research, 39(20),
+              |      5212–5220.
+blank         | 
+ref           | 32. Zhang, G.-S., Qu, J.-H., Liu, H.-J., Liu, R.-P., & Li, G.-T. (2007). Removal
+              |      mechanism of As(III) by a novel Fe-Mn binary oxide adsorbent: Oxidation and
+              |      sorption. Environmental Science & Technology, 41(13), 4613–4619.
+blank         | 
+              | 
+              | 
+              | 
+meta          | 68
+title         | Chapter 4: Distributed microbially- and
+              | chemically-mediated redox processes
+              | controlling arsenic dynamics within Mn-/Fe-
+              | oxide constructed aggregates
+blank         | 
+title         | 4.1 Abstract
+text          |         The aggregate-based structure of soils imparts physical heterogeneity that that
+              | gives rise to variation in microbial and chemical processes that may influence the
+              | speciation and retention of trace elements such as As.      To examine the impact of
+              | distributed redox conditions on the fate of As in soils systems, we imposed various redox
+              | treatments upon constructed soil aggregates composed of ferrihydrite- and birnessite-
+              | coated sands presorbed with As(V) and inoculation with the dissimilatory metal reducing
+              | bacterium Shewanella sp. ANA-3. Aeration of the advecting solution surrounding the
+              | aggregates was varied to simulate environmental conditions. We find that diffusion-
+              | limited transport allows reducing conditions to persist in the interior of the aggregate
+              | when aerated treatments are imposed, causing As, Mn, and Fe to migrate from the
+              | reduced aggregate interiors and become immobilized at the aerated exterior region. Upon
+              | transition to anoxic conditions, pulses of As, Mn and Fe are released into the advecting
+              | solution outside of the aggregate in order of energetic yield coupled with lactate
+              | oxidation during microbial respiration. Inversely, release of reduced species from the
+              | aggregate into the advecting solution is inhibited upon transition from aerated to anoxic
+              | conditions, where the oxidized exterior acts as an oxidizing adsorbent barrier.
+              | Importantly, we find that As(III) oxidation by birnessite is appreciable only in the
+              | presence of O2, where reductive dissolution of Mn oxides inhibits oxidation under
+              | anaerobic conditions.   Our results demonstrate the importance of considering redox
+              | conditions and the physical complexity of soils in determining the As dynamics, where
+              | redox transitions can either enhance or inhibit As release due to speciation shifts in both
+              | sorbents (solubilization versus precipitation of Fe and Mn oxides) and sorbates.
+blank         | 
+              | 
+title         | 4.2 Introduction
+text          |          Arsenic is a ubiquitous contaminant that jeopardizes water quality as a result of
+              | both natural and anthropogenic sources (1). The mobility of As through soils, and
+              | eventual contribution to surface or groundwater, is controlled by biological, chemical,
+              | and physical processes (most notably that influence the oxidation state of As) that are
+              | heterogeneously distributed within surface and subsurface environments. Within soil and
+              | water systems at circumneutral pH, As(V) predominates under aerated, oxidizing
+              | conditions as the oxyanion HxAsO4x-1, while arsenite, as H3AsO30, typically dominates
+              | under anaerobic, reducing conditions. Arsenic(III), though binding extensively to iron
+              | oxides, is generally considered the more mobile species of As (2), while As(V) is less
+              | selective and adsorbs appreciably onto a variety of metal oxyhydroxides, hydroxides, and
+              | oxides (here collectively referred to as oxides) including Fe, Al, and Mn oxides (3-5).
+              |          In many environments, including seasonally saturated soils (6), bioturbated
+              | sediments (7), and forest soils (8), temporary O2 depletion within soil aggregates results
+              | from redox fluctuations (9, 10), where rapid switches in dominant metabolic processes
+              | may occur (7). Iron and Mn oxides are produced by oxidative precipitation under aerated
+              | conditions, while reductive dissolution reproduces Fe(II) and Mn(II) under anaerobic
+              | conditions. The relative flux of Mn and Fe out of soils and sediments as a consequence
+              | of reductive dissolution varies depending on biogeochemical pathways active in a certain
+              | environment (11). Hence, microbial respiration upon Fe and Mn oxides may greatly
+              | impact the transport of adsorbed trace metals including arsenic.
+              |          Physical heterogeneity influences the extent and spatial distribution of oxidative
+              | and reductive processes within soils and sediments.                Soils are composed of
+              | microaggregates fused together by labile organic matter into macroaggregates (12, 13),
+blank         | 
+meta          | 70
+text          | which form a complex matrix of transport mechanisms comprised of advective flow
+              | channels between aggregates combined with diffusion-controlled intra-aggregate
+              | transport (14-16). The rate of intra-aggregate transport of chemical species such as
+              | oxygen from the aggregate exterior decreases toward the aggregate center due to
+              | diminishing pore size, increased tortuosity, and discontinuities (16). Oxygen is further
+              | limited within aggregates through microbial respiration, becoming depleted within
+              | millimeters of the aggregate exterior (10, 17). Depletion of oxygen initiates microbial
+              | anaerobic respiration alternative terminal electron acceptors, including As(V) and Fe(III)
+              | and Mn(IV) oxides common to soil aggregates (18).
+              |          Reductive dissolution and transformation of Fe and Mn oxides and As(V) have
+              | been identified as the primary mechanisms controlling As mobilization within soils (1).
+              | Anaerobic conditions in subsurface soils induce respiration of metal oxides and As(V),
+              | ultimately leading to the release of arsenic into the aqueous phase as the surface area of
+              | the Fe(III) oxides decreases during reductive transformation (3). Microbial reduction of
+              | ferrihydrite initially results in sequestration of As concomitant with transformation to
+              | magnetite; however, continued Fe reduction eventually prompts Fe oxide dissolution and
+              | As release (3, 19).
+              |          Manganese is a highly redox active element in natural systems. In its oxidized
+              | forms (MnIII/IV) is serves as one of nature’s strongest oxidants and a potent adsorbent of
+              | many trace metals (20). Higher valent forms of manganese can undergo dissimilatory
+              | reduction to Mn(II) under anaerobic conditions, while Mn(II), which is kinetically
+              | stabilized towards oxidation at circumneutral pH, is oxidized by molecular oxygen via
+              | mineral surface or bacterial catalysis (21).
+              |          Here, we examine the combined effects of redox oscillations and physical
+              | heterogeneity on transport and transformation of As.         Using synthetic aggregates
+              | composed of birnessite- and ferrihydrite-coated quartz sands presorbed with As(V), we
+              | examined the effects of redox fluctuations on the mobilization and speciation of As
+              | within a chemically and physically complex system. The aggregates were inoculated
+              | with dissimilatory metal reducing bacteria Shewanella sp. ANA-3, capable of respiring
+              | upon As(V), Fe(III), and Mn(IV) and placed in aerated, anoxic, and redox transitioning
+meta          |                                                                                        71
+text          | environments. We reveal that As release rates and concentrations from the aggregates are
+              | highly similar even under different aeration/redox treatments, where As, Fe, and Mn
+              | redox cycling is only active within the outer 3 millimeters of the aggregate exterior.
+              | Under aerated conditions, the exterior of the aggregate remains oxic, forming an Fe(III)
+              | oxide rich rind proximal to advective flow channel that adsorbs and accumulates As.
+              | When the aerated aggregate is transitioned to anoxic conditions, microbial respiration of
+              | Fe(III) oxides and As(V) cause an immediate pulse of As(III) to be released from the
+              | aggregate. In the presence of aeration of advective flow path external of the aggregate,
+              | Fe migrates from the interior and accumulates at the exterior relative to initial
+              | concentrations. However, Mn(II) elution occurs independent of aeration status, albeit to
+              | a lesser extent in the presence of oxygen. Manganese(II) released from the aggregate
+              | occurs prior to Fe(II) release, following thermodynamic favorability of microbial electron
+              | acceptors.
+blank         | 
+              | 
+title         | 4.3 Materials and Methods
+blank         | 
+title         | 4.3.1 Aggregate construction and reactor setup.
+text          | Birnessite was synthesized by dissolving 63 g of KMnO4 in 1 L of doubly deionized
+              | (DDI) water. The solution was heated to 90˚C and combined with 66 mL concentrated
+              | HCl in a separate 4 L flask while being vigorously stirred. The reaction continued at
+              | 90˚C for 10 min, then cooled for 30 min before filtering through a vacuum filtration
+              | system.      Oxides captured by the filter were resuspended in DDI water and filtered
+              | repeated to remove entrained KMnO4. 2-line ferrihydrite was synthesized following
+              | protocol previously outlined by Schwertmann and Cornell, 2000 (22). A small portion of
+              | the birnessite and ferrihydrite was dried and crushed using a mortar and pestle to
+              | confirmed oxide identity by powder X-ray diffraction analysis using Cu K' radiation.
+              |           In separate containers, birnessite and ferrihydrite pastes were mixed with quartz
+              | sand, allowed to air-dry over 2 d, then rinsed repeatedly with DDI water and air-dried for
+              | another 2 d. Birnessite- and ferrihydrite-coated sands were combined to form a 1:10
+              | Mn:Fe molar ratio mixture. Oxide coated sands were sterilized by autoclaving 250 g of
+meta          | 72
+text          | sand in 1 L of DDI water. Phosphate was presorbed to sands by decanting DDI water and
+              | incubating with 0.26 µM NaH2PO4 in 1 L of autoclaved basal salts medium, BSM (10
+              | mM PIPES, 2.7 mM KCl, 0.3 mM MgSO4, 7.9 mM NaCl and 0.4 mM CaCl2.2H2O, and
+              | its pH was adjusted to 7.1 with 3 M HCl) and allowed to incubate at room temperature
+              | for 3 d. Phosphate and BSM was decanted and replaced with 2.5 mM Na2HAsO4(7H2O,
+              | incubated also at room temperature for 3 d, then decanted and sands were rinsed twice
+              | with 250 mL of autoclaved BSM. Arsenate concentration adsorbed to the sands at
+              | experiment intiation was 0.0236 moles As(V)/mole Fe or 0.607 moles As(V)/mole Mn.
+              |          Shewanella sp. ANA-3 was grown aerobically in autoclaved tryptic soy broth
+              | (30 g L-1 DDI water) at 30°C until late log phase from frozen seed culture (stored in 20%
+              | glycerol at -80°C) in 200 mL of solution. Cells were harvested and washed by
+              | centrifuging liquid cultures (5000 x g; 15 min; 25°C) and re-suspended in 30 mL of BSM
+              | at pH 7.1 three times.
+              |          250 g of As(V)-presorbed oxide-coated sand was inoculated with ~8 x 108 cells
+              | g-1 sand, combined with 0.25% agarose (0.25 g UltraPure agarose dissolved in 100 mL
+              | DDI water), and mixed thoroughly to ensure homogeneous distribution of bacteria and
+              | agarose. The bacteria inoculated agarose sand mixture was poured into sterilized molds
+              | to form 2.5 cm diameter spheres. The shaped aggregate had a dry bulk density of 1.21 g
+              | cm-3 and porosity of 0.58.
+blank         | 
+title         | 4.3.2 Flow-through reactor experimental procedure.
+text          |          Shaped aggregates were placed in the center of flow-through reactor made of
+              | polycarbonate (3.7 cm height, 5.1 cm internal diameter) with 0.2 µm filters placed at the
+              | inlet (bottom) and outlet (top) of reactor. A total of eight reactors were prepared where
+              | four were run under aerated-flow conditions and four run under anoxic-flow. All reactors
+              | were initiated with in-flow of BSM amended with 3 mM lactate, 17.8 µM NH4Cl and 1
+              | mL L-1 Wolfe’s mineral solution from bottom of reactors at 1 mL h-1 flow-rate. Aerated-
+              | flow reactors were run on bench-top with filtered air continuously purging solution
+              | surrounding aggregates.      Anoxic-flow reactor experiments were carried out in an
+blank         | 
+              | 
+meta          |                                                                                       73
+text          | anaerobic glove-bag in a 95% N2:5% H2 atmosphere. Effluent was collected from the
+              | outlet (top) of reactors at 1 mL h-1.
+blank         | 
+title         | 4.3.3 Aqueous phase analysis.
+text          |          Aqueous As, Mn, and Fe concentrations were measured in filtered effluent
+              | samples using inductively coupled plasma optical emission spectrometry (ICP-OES).
+              | The lower detection limits for measuring As, Mn, and Fe were 5, 1, and 18 µg L-1,
+              | respsectively. Another 3 mL of filtrate was used for As(III)/As(V) speciation following
+              | the method of Masscheleyn et al. (1991) as modified by Jones et al. (2000) as follows:
+              | while purging with N2, 0.6 mL of 2 M Tris (pH 6.0) was added to 3 mL of sample; after
+              | Tris is thoroughly mixed into sample, two additions of 0.3 mL of 3% (w/v) NaBH4 in 1
+              | M NaOH is added to sample tube, with 5 min of N2 purging between additions. Lactate
+              | and acetate concentrations were determined from1 mL of filtrate stored at -20°C after
+              | sampling using ion chromatography.
+blank         | 
+title         | 4.3.4 Solid phase analyses.
+text          |          Aggregates were broken down for solid phases analysis after 48 d of flow. Each
+              | aggregate was separated into three concentric zones labeled as ‘E’ for exterior (0 to 3.5
+              | mm), ‘M’ for midsection (3.5 to 7.5 mm), and ‘I’ for interior (7.5 to 12.5 mm). Sands
+              | from each zone were dried and used for bulk X-ray absorption spectroscopic (XAS)
+              | analysis including, X-ray absorption near-edge structure (XANES) spectral collection to
+              | determine ratio of As(III) and As(V), Fe extended X-ray absorption fine structure
+              | (EXAFS) spectroscopy to quantify Fe phases, and acid digestion with 6 M HCl for
+              | quantifying solid phase As, Fe, and Mn concentrations. Triplicate sand digestions were
+              | averaged to determine initial solid phase As (17.054±1.23 mmol kg-1 sand), Fe
+              | (709.562±27.02 mmol kg-1 sand), and Mn (35.462±6.60 mmol kg-1 sand) concentrations.
+              |          Bulk XAS was conducted on beamlines 11-2 and beamline 4-1 at Stanford
+              | Synchrotron Radiation Laboratory (SSRL) using method described previously (Masue-
+              | Slowey et al., 2011).       Dried sands from each aggregate section were sonicated
+              | anaerobically in DDI water. Homogenous As-Fe-Mn layers were collected by vacuum
+meta          | 74
+text          | filtration of aqueous phase from sonicated samples on cellulose nitrate filters and sealed
+              | between Kapton tape.        Double-crystal, Si(220) monochromators were used at both
+              | beamlines for energy selection. Fe EXAFS spectra were obtained from 100 eV below to
+              | 1000 eV above the Fe K-edge at 7111 eV. Fe solid-phase speciation was quantified by
+              | performing linear combination fitting on Fe EXAFS collected on bulk samples with k3-
+              | weighted EXAFS spectra of Fe standard compounds using the SIXPACK interface to
+              | IFFEFIT (Webb, 2005). Iron fluorescence spectra were normalized and backscattering
+              | contribution isolated by spline function subtraction.      Normalized data (eV) were
+              | converted to k-space (Å-1), and k3 weighted. Linear-combination fitting was performed
+              | from 3 to 14 Å-1 and results were evaluated based on reduced X2 values. Ferrihydrite,
+              | magnetite, and goethite were chosen as reference compounds for fittings based on
+              | reaction products reported in similar past studies (19), and phases identified using
+              | scanning electron microscopy (SEM).         Arsenic speciation of bulk samples were
+              | determined by analyzing the near-edge portion of the As spectra collected from 240 eV
+              | below to 430 eV above the As(V) K-edge at 11874 eV. Ratio of As(III) and As(V)
+              | adsorbed to solid phase samples were determined with linear-combination fitting of
+              | normalized XANES spectra with spectra collected for As(III) and As(V)-sorbed
+              | ferrihydrite as fitting standards.
+              |          Micro-X-ray fluorescence (µ-XRF) analysis of radial slices of each aggregate
+              | was carried out at beamline 2-3 at SSRL and beamline 10.3.2 at ALS to map the spatial
+              | distribution of As(III), As(V), Fe, and Mn from the exterior to interior of aggregates.
+              | Aggregate slices were dried in anaerobic glove-bag, embedded in EPOTEK301-2FL
+              | epoxy, thin-sectioned to 30 µm thickness and mounted on a quartz slide. Maps were
+              | taken at three energies (11871, 11874, and 11880 eV) at 6 to 10 µm step size for low-
+              | resolution maps and 2 to 5 µm step size for high-resolution maps. Arsenic µ-XANES
+              | points were chosen using µ-XRF maps and analyzed for As(V)/As(III) ratio at each
+              | location using the same analysis technique described for As speciation on bulk samples.
+              | Arsenic speciation across the aggregates was determined by the XANES imaging
+              | subroutine SMAK—a subroutine of SIXPAK (23).
+blank         | 
+              | 
+meta          |                                                                                        75
+title         | 4.4 Results
+blank         | 
+title         | 4.4.1 Aqueous phase results from aggregate reactors.
+text          |          Under aerated conditions, effluent As(V) is an initial concentration >50 µM but
+              | undergoes rapid decay to a pseudo-steady-state concentration of ~2 µM decreased below
+              | As(III) concentration after 2 d of reaction (Figure 4.1A). Arsenic(III) concentration is
+              | initially lower than that of As(V), but over the first ~25 d undergoes a more gradual
+              | decrease then becoming the dominant form of As in the effluent from 2 to 23 d of
+              | reaction; after 25 d of elution, its concentration decreases below our level of detection
+              | (Figure 4.1A). Overall, a greater mass of As(III) was eluted from the reactor than As(V),
+              | with a total of 8.26 and 4.57 µmol removed, respectively. Effluent Mn(II) concentrations
+              | increase from 2.7 to 130 µM after 6 d of reaction followed by gradual decreased to 40
+              | µM over the remainder of the reaction period (Figure 4.1B). Aqueous Fe(II) remained at
+              | or below the detection limit in the presence of oxygen (Figure 4.1B).
+              |          Under anoxic advecting solute conditions, As(III) was the dominant As species
+              | from the first sampling, with As(V) near or below detection limit throughout the
+              | experiment (Figure 4.2A). Manganese(II) concentrations peaked after 6 d of reaction, as
+              | seen under aerated treatment; however, the maximum concentration of Mn(II) measured
+              | was nearly three folds greater (372 µM) than the maximum under aerated conditions (126
+              | µM) (Figure 4.2B). Effluent Mn(II) concentrations began to decrease after 7 d, with
+              | concentrations reaching detection limit at approximately 30 d of reaction. Absence of
+blank         | 
+              | 
+              | 
+              | 
+meta          | 76
+text          | Figure 4.1 Dissolved As (A) and Fe and Mn (B) in effluent from aerated reactor. Dissolved total
+              | As (black dots), As(III) (open squares), and As(V) (black squares), Fe (white circles), and Mn
+              | (black circles) are shown.
+              | Figure 4.2 Dissolved As (A) and Fe and Mn (B) in effluent from anoxic reactor. Dissolved total
+              | As (black dots), As(III) (open squares), and As(V) (black squares), Fe (white circles), and Mn
+              | (black circles) are shown.
+blank         | 
+              | 
+              | 
+meta          | 78
+text          | abiotic oxidation, Fe(II) concentrations increased over the first 15 d and stabilizing at
+              | approximately 180 µM for the remainder of the reaction period (Figure 4.2B).
+              |          When anoxic reactors are transitioned to oxic conditions in the advecting
+              | solution after a 20 d reaction period, As(III) concentrations dominated over As(V) in the
+              | effluent throughout the experimental period (Figure 4.3A). Total As concentrations
+              | gradually decreased from 54 µM to 18 µM over the first 17 d of anoxic solution addition;
+              | upon switching to aerated conditions, a decrease in total effluent As concentration from
+              | 18 µM to 13 µM (Figure 4.3A) results. Response to the aeration transition was also
+              | reflected in Fe(II) concentrations, where effluent Fe(II) concentrations peaked just before
+              | the switch to aerated conditions, where upon Fe decreased rapidly to our level of
+              | detection.   Manganese(II) concentrations are less affected by transition to aerated
+              | conditions owing to effective depletion of Mn in the eluting solution over the first 20 d—
+              | a maximum concentration occurs after 6 d of reaction (consistent under all aeration
+              | treatments) and then undergoes progressive decay for the next 14 d.
+              |          For aggregates first subjected to aerated advecting solutions, a switch to
+              | anaerobic conditions after 20 d leads to a nearly immediate pulse (2 d) of As(III), a
+              | rebound in Mn(II) concentration, and a progressive increase in Fe(II) within effluent
+              | solutions. Similar to results from the continually aerated aggregate reactor, during the
+              | aeration period As(III) concentrations increase to a maximum of %30 uM within the first
+              | 5 d and then progressively decreases until the cessation of aeration at day 20. A pulse of
+              | As(III) (increase from 10 to 16 µmol L-1) occurred 2 d after the transition to anoxic
+              | advecting solution conditions. Similarly, Mn(II) concentrations decrease from 200 µM
+              | on day 6 to 107 µM over 11 d under aerated advecting solution conditions; upon the
+              | transition to anoxic exterior conditions, a second pulse of Mn(II) was released, reaching a
+              | comparable magnitude (210 µM) as the first concentration peak and then decaying to 40
+              | µM over the following 11 d. Iron(II) concentrations were below detection limit under
+              | aerated conditions (20 d), and then increased rapidly to 201 µM 5 d after the transition to
+              | anoxic exterior conditions, reaching a maximum concentration of 225 µM.
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                         79
+text          | Figure 4.3 Dissolved As (A) and Fe and Mn (B) in effluent from anoxic-to-aerated transition
+              | reactor. Dissolved total As (black dots), As(III) (open squares), and As(V) (black squares), Fe
+              | (white circles), and Mn (black circles) are shown.
+blank         | 
+              | 
+              | 
+              | 
+meta          | 80
+text          | Figure 4.4 Dissolved As (A) and Fe and Mn (B) in effluent from aerated-to-anoxic transition
+              | reactor. Dissolved total As (black dots), As(III) (open squares), and As(V) (black squares), Fe
+              | (white circles), and Mn (black circles) are shown.
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                                   81
+text          | Figure 4.5 Fraction of final concentration relative to initial for mass of As (gray), Fe (black), and
+              | Mn (white) in the exterior (E), mid (M), and interior (I) section for aerated (A), anoxic (B),
+              | aerated-to-anoxic transitioned (C), and anoxic-to-aerated transitioned (D) aggregates.
+blank         | 
+title         | 4.4.2 Solid phases analysis.
+text          |           Redistribution of As, Fe, and Mn content and speciation varied between
+              | aggregates under differing redox treatments.             Consistent with findings reported by
+              | Masue-Slowey et al. (17), total solid-phase Fe content of aggregate exteriors increased by
+              | 49 µmol relative to the initial concentration over 47 d of reaction under aerated
+              | conditions, while the mid-section and interior regions lost 30 and 7.7 µmol of Fe (Table
+              | 4.1). Similarly, the highest Fe mass accumulated in the exterior region (only 14 µmol Fe
+              | decrease from initial mass) and was depleted from interior regions of anoxic to aerated
+              | transitioned aggregate. Anoxic and aerated-to-anoxic transitioned aggregates both lost Fe
+              | from all regions relative to the initial mass (Table 4.1 and Figure 4.5). Iron EXAFS
+              | analyses revealed that ferrihydrite was the dominant (>89 mol-%) Fe solid phase in all
+              | aggregates,
+blank         | 
+              | 
+meta          | 82
+text          | Table 4.1 Solid phase characteristics of aggregate sections
+blank         | 
+text          |                     Total     Total    Total     As/Fe    As/Mn           As           Fe             Mn
+              |                     As        Fe       Mn
+              |                                                  molar    molar       relative     relative         relative
+              |                     (µmol)    (µmol)   (µmol)
+              |                                                   ratio       ratio   to initial   to initial   to initial
+              | Initial     E       36.3      446      17.4     0.0238    0.609       -            -            -
+              |             M       17.8      219      8.56     0.0237    0.607       -            -            -
+              |             I       3.57      43.9     1.712    0.0237    0.607       -            -            -
+              |             Total   57.7      709      27.7     0.0236    0.607       -            -            -
+blank         | 
+              | 
+text          | Aerated     E       28.1      495      9.54     0.0568    2.95        0.775        1.11         0.548
+              |             M       13.5      189      0.73     0.0714    18.5        0.759        0.862        0.0852
+              |             I       3.35      36.2     0.81     0.0922    4.12        0.937        0.827        0.0474
+              |             Total   45.0      720      11.1     0.0624    4.06        0.780        1.02         0.400
+blank         | 
+              | 
+text          | Anoxic      E       27.5      379      0.229    0.0727    120         0.759        0.849        0.0132
+              |             M       15.8      178      0.120    0.0888    131         0.886        0.809        0.0140
+              |             I       3.38      35.7     0.028    0.0947    121         0.947        0.813        0.0163
+              |             Total   46.7      592      0.377    0.0789    124         0.810        0.834        0.0136
+blank         | 
+              | 
+text          | Aer->Ano    E       23.7      372      0.307    0.0638    77.3        0.654        0.833        0.0176
+              |             M       15.3      174      0.167    0.0880    91.8        0.861        0.794        0.0195
+              |             I       3.45      34.5     0.044    0.100     78.4        0.967        0.7856       0.0257
+              |             Total   42.5      580      0.518    0.0732    82.1        0.737        0.818        0.0187
+blank         | 
+              | 
+text          | Ano->Aer    E       28.5      432      2.01     0.0660    14.2        0.786        0.969        0.116
+              |             M       16.1      178      0.213    0.0908    75.7        0.906        0.809        0.0249
+              |             I       3.37      35.5     0.135    0.0950    25.0        0.944        0.809        0.0789
+              |             Total   48.0      645      2.36     0.0744    20.3        0.833        0.910        0.0853
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                                                83
+text          | Figure 4.6 X-ray fluorescence mapping of Fe and Mn (top panel 1) and As(III) and As(V)
+              | (bottom panel 2) in (A) aerated, (B) anoxic, (C) anoxic-to-aerated, (D) aerated-to-anoxic
+              | aggregates.
+blank         | 
+text          | while magnetite composed a smaller portion (7-11 mol-%) and was evenly distributed
+              | across the three regions of the aggregate (Table 4.2).
+              |          The greatest mass of total solid-phase As was lost from the exterior sections
+              | while higher amounts were maintained in interior regions of all aggregates (Table 4.1 and
+              | Figure 4.5). Between 21 to 35 % of As was lost from the exterior, 9.4 to 24 % from the
+              | mid-section, and only 3.3 to 6.3 % from the interior regions. µ-X-ray fluorescence (µ-
+              | XRF) mapping and µ-XANES analysis (Figures 4.6 and 4.7) illustrated that As(V) was
+              | present in the exterior of aerated and anoxic-to-aerated aggregates only (Figure 4.7);
+              | As(V) was the dominant As species in the outer 2 mm of the aggregate. A transition zone
+blank         | 
+              | 
+              | 
+meta          | 84
+text          | Table 4.2 Solid phase As and Fe speciation in aggregate sections
+blank         | 
+text          |                                As speciation (mol %)           dominant Fe mineralogy (mol %)
+              |                               As(V)            As(III)             ferrihydrite   magnetite
+              | Oxic             E              46               54                    93             7
+              |                  M              15               85                    90             10
+              |                  I              10               90                    91             9
+blank         | 
+              | 
+text          | Anoxic           E               9               92                    92             8
+              |                  M              16               84                    91             9
+              |                  I              19               81                    92             8
+blank         | 
+              | 
+text          | Ox->Ano          E              17               83                    93             7
+              |                  M              15               85                    89             11
+              |                  I              12               88                    90             10
+blank         | 
+              | 
+text          | Ano->Ox          E              29               71                    92             8
+              |                  M              28               72                    89             11
+              |                  I              33               67                    93             7
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                            85
+text          | Figure 4.7 As species distribution for aggregate cross section determined by As !-XANES
+              | analysis from exterior (E), mid (M), to interior (I) sections of (A) aerated, (B) anoxic, (C) aerated-
+              | to-anoxic, (D) anoxic-to-aerated.
+blank         | 
+text          | occurs from 2 to 5 mm into the aggregates where the proportion of As(III) increases and
+              | then dominates (~80 % As(III)) the solid phase speciation (Figure 4.7). By contrast, 70-
+              | 80 % As(III) and only a maxiumum of 20-30 % As(V) is found in anoxic and oxic-to-
+              | anoxic transition aggregates from the exterior to interior regions (Figure 4.7).
+              |           Total solid-phase Mn was greatly depleted (loss of >89 mol-% of initial mass)
+              | from all sections of the aggregates except under aerated conditions, which retained 55,
+              | 8.5, and 4.7 % Mn in the exterior, mid-section, and interior of the aggregate, respectively,
+              | similar to the increased mass near the exterior seen in the redistribution of Fe (Table 4.1).
+              | µ-XRF mapping of aerated aggregate confirms higher concentration of Mn near the
+              | exterior of the aggregate and absence of Mn signal as distance from exterior increases
+              | (Figure 4.6).    Manganese redistribution in anoxic-to-aerated aggregate mirrored that of
+blank         | 
+meta          | 86
+text          | the aerated aggregate, with 11.6 % remaining in the exterior, and much less, 2 – 8 % left
+              | in the mid and interior sections. Anoxic and aerated-to-anoxic aggregates lost slightly
+              | more from the exterior sections than other sections, with losses of 97.4 and 98.4 % from
+              | the interior and 98.7 and 98.2 % from the exterior.
+blank         | 
+              | 
+title         | 4.5 Discussion
+text          |          Reduction-oxidation (redox) transitions in soils lead to the redistribution of
+              | metal oxides and sorbed trace metals. We examined the effects of prolonged aerated and
+              | anoxic conditions on synthetic aggregates composed of As(V) presorbed ferrihydrite- and
+              | birnessite coated sands inoculated with dissimilatory metal reducing bacteria, Shewanella
+              | sp. ANA-3, which are capable of respiring upon all three metals and oxygen while
+              | utilizing lactate as a carbon and electron source. Furthermore, a comparison is made
+              | between aggregates that undergo single aeration treatment (i.e. continuously anoxic or
+              | aerated conditions) and aeration status transitioned aggregates, which were maintained
+              | under aerated or anoxic conditions for 20 days then switched to anoxic or aerated
+              | environments for another 30 days.
+              |          Spatial redistribution and speciation of As, Fe, and Mn within the aggregates are
+              | controlled by redox gradients resulting from progressively decreasing oxygen
+              | concentrations as one approaches the aggregate interior. As was clearly illustrated by
+              | Masue-Slowey et al. (17), that despite having ample oxygen supplied at the aggregate
+              | exterior under aerated conditions, anaerobic conditions arise within millimeters of the
+              | exterior due to greater rate of oxygen respiration by Shewanella than rate of oxygen
+              | diffusion into the aggregate. Hence, for elements such as Fe and Mn that have more
+              | mobile reduced, biogeochemical conditions of aggregate interior leads to mobilization
+              | and diffusion toward the exterior.     When the advecting solution is aerated, Fe(II)
+              | produced within the aggregate interior diffuses toward the exterior where upon it
+              | undergoes oxidation.     Subsequent precipitation of Fe(III) oxide results in the co-
+              | association of As, which also diffuses from the reducing aggregate interior toward the
+              | exterior. Upon reaching the metal oxide rind, As(III) is oxidized to As(V) by residual
+blank         | 
+meta          |                                                                                        87
+text          | Mn oxides, producing a mixture of As(V) and As(III) associated with the aerated
+              | aggregate exterior (Figures 4.6 and 4.7). Interestingly, As redistribution within the
+              | aggregates was highly similar between all aeration treatments (Figure 4.5), with
+              | increasing As/Fe ratios approaching the interior indicative of As retention concomitant
+              | with release of Fe. Microbial respiration of ferrihydrite is active in the anaerobic center of
+              | the aggregate to comparable levels to those exposed to anoxic advecting solutes, leading
+              | to production of Fe(II). These results demonstrate that influence of oxygen (or aeration
+              | status) on As dynamics is most pronounced in the exterior section, whereas reducing
+              | conditions in the interior are similar across various aeration conditions.
+              |          Arsenic that is accumulated in the Fe oxide exterior under aerated conditions
+              | was predicted to mobilize under anaerobic conditions (18). Our results support this
+              | hypothesis, where As concentrations in the effluent of aerated-to-anoxic aggregate
+              | gradually decreased over the first 20 days of aeration being composed of a mixture of
+              | As(V) and As(III). When anoxic conditions were imposed upon the aerated aggregate on
+              | day 20, a pulse of As was released and As(V) concentrations in the effluent decreased
+              | (Figure 4.4B). Manganese and Fe were also measured in the effluent, though their peak
+              | concentrations were delayed by 4 and 13 d, respectively, compared to peak As
+              | concentration. Therefore, as the redox status of the exterior environment (advecting
+              | solution) shifts from aerobic to anaerobic, an immediate pulse of As release occurs which
+              | is then followed by Mn and Fe.      Multiple studies support the release of As independent
+              | of Fe reduction, consistent with our finding that Fe elution is not a prerequisite for As
+              | release from the constructed aggregates (17, 19, 24). Reverting to aerated conditions
+              | after anoxic treatment slowly decreases the rate of As release, as demonstrated by the
+              | decrease in total As eluted after aeration onset, and As(V) re-appears in the effluent
+              | concomitant with Fe oxidation and removal from effluent (Figure 4.2A and 4.4A).
+              |          Regardless of aeration status, As(III) composes approximately 80 mol-% of the
+              | total solid phase As within the aggregate at distances greater than ~3 mm from the
+              | advective flow channel.      Therefore, As redox cycling occurs predominantly in the
+              | exterior region of the aggregate, while mid and interior sections are highly reducing. The
+              | predominantly As(III)-containing interior shows that As(III) oxidation by Mn oxides is
+meta          | 88
+text          | minimal and likely only occurs transiently prior to anoxic onset within the aggregate
+              | interior. Conditions that promote Mn reduction inhibits appreciable As(III) oxidation due
+              | to reductive dissolution of Mn oxides; our results demonstrate that the presence of O2 is
+              | required for Mn oxides to be an effective oxidant of As(III) under conditions operative
+              | for dissimilatory reduction of Mn. Similarly, Tokunaga et al. (14) demonstrated that
+              | immediately following carbon amendment to sediments containing U(VI) and Fe and Mn
+              | oxides, rapid and complete reduction of Mn(III,IV) oxides to Mn(II) was observed
+              | indicating Mn oxides were not involved in U(IV) oxidation after dissolution. Instead,
+              | As(III) oxidation by Mn oxides likely takes place in the aggregate exterior regions, where
+              | As(III) diffused from the interior can be rapidly oxidized by Mn oxides and subsequently
+              | adsorbed to the surrounding Fe oxide matrix.
+              |          Our results illustrate that oxic and anoxic cycling of soils will alter the retention
+              | and distribution of trace elements such as arsenic. Owing to mass transfer limited O2
+              | supply and sustained microbial activity, only the exterior region (outer few millimeters)
+              | of soil aggregates may be aerobic even under seemingly well aerated conditions. Owing
+              | the diffusive gradients established from the anaerobic aggregate interior and aerobic
+              | exterior, redox active elements such as Fe, Mn, and As, all having generally more
+              | soluble/mobile species under reducing conditions, will migrated and build in
+              | concentration at aggregate exteriors.      Upon a transition from aerobic to anaerobic
+              | conditions, the accumulated elements will be released to the aqueous phase and result in a
+              | concentration pulse within advecting waters. However, the effluent profiles for Mn and
+              | Fe differ considerably. Respiration on Mn oxides is thermodynamically more favorable
+              | than reduction of Fe oxides when coupled with most carbon sources (18).               Hence,
+              | sediment profiles containing both Mn and Fe oxides generally exhibit clearly stratified
+              | redox layers where Mn reduction occurs at shallower depths than Fe reduction (25).
+              | Similarly, Mn(II) elution from aggregates under anoxic conditions occurred prior to
+              | Fe(II) elution. Manganese flux from the aggregates occurred under both aerated and
+              | anoxic conditions due to the large activation energy required to oxidize Mn(II) and the
+              | requirement for a microbial or mineral catalyst (21, 26, 27); in contrast, abiotic Fe(II)
+              | oxidation by molecular oxygen is rapid (28), making Fe flux out of aggregates much
+meta          |                                                                                            89
+text          | more dependent upon aeration status—with aeration resulting in a build-up of Fe in
+              | aggregate exteriors and limited release to groundwater. Certain trace metals have been
+              | found to be more strictly associated with Mn oxides versus Fe oxides; temporal variations
+              | in trace metal efflux from soils and sediments may, therefore, be caused by the
+              | differential reduction of Mn and Fe oxides over time and space.
+blank         | 
+              | 
+title         | 4.6 Conclusions
+text          |          Fate and transport of As, Fe, and Mn is influenced by both redox transitions and
+              | the physical heterogeneity inherent to soils that results in a distribution of biogeochemical
+              | conditions. The structure of soil aggregates allows reductive processes to persist within
+              | the aggregate interiors even under seemingly aerated conditions, with oxidizing processes
+              | being restricted to only a few millimeters of aggregate exteriors. Therefore, reductive
+              | dissolution of As, Mn, and Fe (and reduction of a large suite of constituents) actively
+              | occur regardless of oxygenation within advective flow channels surrounding soil
+              | aggregate. However, release of reduced species from aggregates is highly dependent
+              | upon the metal and aeration status; Mn elution occurs prior to the release of Fe and can
+              | be operative under aerated or anoxic conditions, while Fe is only released out of the
+              | aggregate when the Fe oxide rind is reduced. However, rapid Mn(II) oxidation by
+              | Mn(II)-oxidizing microorganisms has been observed in many oxic environments (29), a
+              | process that can inhibit Mn elution from aggregates in a similar fashion to abiotic
+              | oxidation of Fe(II). Interestingly, As elution from the aggregate is continuous and
+              | comparable under aerated and anoxic conditions; however, As accumulated on the
+              | aggregate exterior (due to adsorption onto Fe oxides formed the oxidation from Fe(II)
+              | diffused from aggregate interior), is released when aerated flow channels transition to
+              | anoxic conditions, producing a sudden high concentration discharge of As into the
+              | advective flow channel. Collectively, our results demonstrate the importance of soil
+              | structure and redox transitions when assessing the mobilization, speciation, and flux of
+              | metal species from soils and sediments.
+blank         | 
+              | 
+              | 
+meta          | 90
+title         | 4.7 Acknowledgements
+text          |         This research was supported by the Stanford NSF Environmental Molecular
+              | Science Institute (NSF-CHE-0431425) and by the National Science Foundation (grant
+              | number EAR- 0952019), and by U.S. EPA STAR graduate fellowship (FP-91678701-3)
+              | to S.C.Y. We thank Guangchao Li for analytical assistance and Sam Webb and Matthew
+              | Marcus for µ-XAS/XRF data acquisition and analysis. We thank Yuji Arai and Chris
+              | Fuller for the suggestion in thin section preparations for µ-XAS/ XRF. Portions of this
+              | research were carried out at SSRL and ALS, user facilities supported by the Department
+              | of Energy, Office of Basic Energy Sciences.
+blank         | 
+              | 
+title         | 4.8 References
+ref           | 1.    Smedley P, Kinniburgh D (2002) A review of the source, behaviour and
+              |       distribution of arsenic in natural waters. Appl Geochem 17:517–568.
+blank         | 
+ref           | 2.    Gupta S, Chen K (1978) Arsenic Removal by Adsorption. J Water Pollut Con F
+              |       50:493–506.
+blank         | 
+ref           | 3.    Tufano KJ, Fendorf S (2008) Confounding impacts of iron reduction on arsenic
+              |       retention. Environ Sci Technol 42:4777–4783.
+blank         | 
+ref           | 4.    Masue Y, Loeppert RH, Kramer TA (2007) Arsenate and arsenite adsorption and
+              |       desorption behavior on coprecipitated aluminum : iron hydroxides. Environ Sci
+              |       Technol 41:837–842.
+blank         | 
+ref           | 5.    Manning B, Fendorf S, Bostick B, Suarez D (2002) Arsenic(III) oxidation and
+              |       arsenic(V) adsorption reactions on synthetic birnessite. Environ Sci Technol
+              |       36:976–981.
+blank         | 
+ref           | 6.    Flessa H, Fischer W (1992) Plant-Induced Changes in the Redox Potentials of Rice
+              |       Rhizospheres. Plant Soil 143:55–60.
+blank         | 
+ref           | 7.    ALLER R (1994) Bioturbation and Remineralization of Sedimentary Organic-
+              |       Matter - Effects of Redox Oscillation. Chem Geol 114:331–345.
+blank         | 
+ref           | 8.    Silver W, Lugo A, Keller M (1999) Soil oxygen availability and biogeochemistry
+              |       along rainfall and topographic gradients in upland wet tropical forest soils.
+              |       Biogeochemistry 44:301–328.
+blank         | 
+              | 
+meta          |                                                                                     91
+ref           | 9.    Fischer Von J (2002) Separating methane production and consumption with a
+              |       field-based isotope pool dilution technique. Global Biogeochem Cycles.
+blank         | 
+ref           | 10.   Sexstone A, Parkin N (1985) Direct Measurement of Oxygen Profiles and
+              |       Denitrification Rates in Soil Aggregates. Soil Science Society of America ….
+blank         | 
+ref           | 11.   Van Cappellen P et al. (1998) Biogeochemical cycles of manganese and iron at the
+              |       oxic-anoxic transition of a stratified marine basin (Orca Basin, Gulf of Mexico).
+              |       Environ Sci Technol 32:2931–2939.
+blank         | 
+ref           | 12.   Jastrow J (1996) Soil aggregate formation and the accrual of particulate and
+              |       mineral-associated organic matter. Soil Biol Biochem 28:665–676.
+blank         | 
+ref           | 13.   Tisdall J, Oades J (1982) Organic matter and water-stable aggregates in soils -
+              |       Tisdall - 2006 - Journal of Soil Science - Wiley Online Library. European Journal
+              |       of Soil Science.
+blank         | 
+ref           | 14.   Tokunaga T et al. (2003) Distribution of chromium contamination and microbial
+              |       activity in soil aggregates. J Environ Qual 32:541–549.
+blank         | 
+ref           | 15.   Vogel HJ, Cousin I, Ippisch O, Bastian P (2006) The dominant role of structure for
+              |       solute transport in soil: experimental evidence and modelling of structure and
+              |       transport in a field experiment. Hydrol Earth Syst Sc 10:495–506.
+blank         | 
+ref           | 16.   Horn R, Taubner H, Wuttke M, Baumgartl T (1994) Soil physical-properties
+              |       related to soil-structure. Soil Till Res 30:187–216.
+blank         | 
+ref           | 17.   Masue-Slowey Y, Kocar BD, Jofre SAB, Mayer KU, Fendorf S (2011) Transport
+              |       Implications Resulting from Internal Redistribution of Arsenic and Iron within
+              |       Constructed Soil Aggregates. Environ Sci Technol 45:582–588.
+blank         | 
+ref           | 18.   Baas-Becking L, Kaplan I, Moore D (1960) Limits of the natural environment in
+              |       terms of pH and oxidation-reduction potential. J Geol 68:243–284.
+blank         | 
+ref           | 19.   Kocar BD, Herbel MJ, Tufano KJ, Fendorf S (2006) Contrasting effects of
+              |       dissimilatory iron(III) and arsenic(V) reduction on arsenic retention and transport.
+              |       Environ Sci Technol 40:6715–6721.
+blank         | 
+ref           | 20.   Post J (1999) Manganese oxide minerals: Crystal structures and economic and
+              |       environmental significance. P Natl Acad Sci Usa 96:3447–3454.
+blank         | 
+ref           | 21.   Diem D, Stumm W (1984) ScienceDirect - Geochimica et Cosmochimica Acta : Is
+              |       dissolved Mn2+ being oxidized by O2 in absence of Mn-bacteria or surface
+              |       catalysts? Geochim Cosmochim Ac.
+blank         | 
+              | 
+              | 
+meta          | 92
+ref           | 22.   Schwertmann U, Cornell RM eds. (2000) Iron Oxides in the Laboratary
+              |       Schwertmann U, Cornell RMeds (Wiley-VCH Verlag GmbH, Weinheim,
+              |       Germany).
+blank         | 
+ref           | 23.   Webb SM (2005) SIXpack: a graphical user interface for XAS analysis using
+              |       IFEFFIT. Phys Scripta T115:1011–1014.
+blank         | 
+ref           | 24.   Campbell KM, Malasarn D, Saltikov CW, Newman DK, Hering JG (2006)
+              |       Simultaneous microbial reduction of iron(III) and arsenic(V) in suspensions of
+              |       hydrous ferric oxide. Environ Sci Technol 40:5950–5955.
+blank         | 
+ref           | 25.   Canfield D, Thamdrup B, Hansen J (1993) the Anaerobic Degradation of Organic-
+              |       Matter in Danish Coastal Sediments - Iron Reduction, Manganese Reduction, and
+              |       Sulfate Reduction Geochim Cosmochim Ac 57:3867–3883.
+blank         | 
+ref           | 26.   Tebo B et al. (2004) Biogenic manganese oxides: Properties and mechanisms of
+              |       formation. Annu Rev Earth Pl Sc 32:287–328.
+blank         | 
+ref           | 27.   Nealson K, Tebo B, Rosson R (1988) Occurrence and Mechanisms of Microbial
+              |       Oxidation of Manganese.. Adv Appl Microbiol 33:279–318.
+blank         | 
+ref           | 28.   Stumm W, Lee GF (1961) Oxygenation of Ferrous Iron. Ind Eng Chem 53:143–
+              |       146.
+blank         | 
+ref           | 29.   Nealson K (2006) in The Prokaryotes, Dworkin M, Falkow S, Rosenberg E,
+              |       Schleifer K-H, Stackebrandt Eeds (Springer New York), pp 222–231. Available at:
+              |       http://dx.doi.org/10.1007/0-387-30745-1_11.
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                   93
+              | 94
+title         | Chapter 5: Analysis of Arsenic(V)-reducing
+              | microbial community structure and
+              | environmental influences using multivariate
+              | statistics
+blank         | 
+title         | 5.1 Abstract
+text          |          Arsenic (As) cycling within soils and sediments of the Mekong Delta of
+              | Cambodia is strongly affected by drastic redox fluctuations caused by seasonal
+              | monsoons. Repeated oxidation and reduction of soils, which contain 10-40 mg kg-1 As,
+              | lead to the eventual downward movement of As to the underlying aquifer. Extensive
+              | flooding during monsoon seasons creates anoxic soil conditions that favor anaerobic
+              | microbial metabolic processes, including arsenate (As(V)) respiration–a process
+              | contributing to the mobilization of As. Amplification of a highly conserved functional
+              | gene encoding dissimilatory As(V) reductase, arrA, can be used as a molecular marker to
+              | detect the genetic potential for As(V) respiration in environmental samples. However,
+              | few studies have successfully amplified arrA without prior enrichment, and factors
+              | influencing sequence diversity are currently unknown. In the present study, we examine
+              | the distribution and diversity of arrA genes amplified from multiple study sites within the
+              | Mekong Delta at various soil depths (10, 50, 100, 200, and 400 cm). Additionally, results
+              | from geochemical analysis of soil porewater collected using lysimeters at the
+              | corresponding depths were applied in multivariate analysis to determine whether
+              | variations in arrA sequence could be explained by environmental factors. Here we report
+              | successful amplification, cloning, and sequencing of 223 novel arrA gene sequences from
+              | Cambodia soils without prior enrichment/stimulation, collectively forming a clade that is
+blank         | 
+meta          |                                                                                         95
+text          | phylogenetically distinct from existing sequences in the database.       Application of
+              | permutational MANOVA demonstrates that As and depth variables are most strongly
+              | associated with variations in arrA sequences, whereas a number of other porewater
+              | constituents (including Fe) appeared insignificant.      Our findings demonstrate the
+              | potential for using biogeochemically and ecologically relevant functional genes to
+              | understand operative geochemical processes and, in particular, conditions promoting the
+              | mobilization of arsenic within flooded, tropical soils—systems where arsenic is having a
+              | devastating impact on human health by contaminating regional aquifers.
+blank         | 
+              | 
+title         | 5.2 Introduction
+text          |         Millions in south and southeast Asia are currently exposed to arsenic (As)
+              | concentrations as high as three orders of magnitude greater than the WHO suggested
+              | limit of 10 µg L-1. Arsenic-bearing minerals derived from the Himalayas is transported
+              | down river channels and deposited into deltas below including the Ganges–Brahmaputra–
+              | Meghna (1, 2), Red River (3), and Mekong River deltas (2, 4, 5). Whereas groundwater
+              | irrigation has been employed for many decades within the Ganges–Brahmaputra–Meghna
+              | basin, groundwater pumping is limited within the Mekong Delta, providing a less
+              | perturbed representation of the As release within southeast Asia. Studies have reported
+              | that, even with relatively minimal groundwater extraction, aquifer As concentrations in
+              | one of the most densely populated area of the Mekong Delta (between the Mekong and
+              | Basaac Rivers) range from 100 to >1000 )g L-1, with an average of ~ 500 )g L-1 (2, 4).
+              | Redox processes within near-surface sediments are responsible for the supply and release
+              | of As into groundwater (2, 4).     Oxidation of Himalayan-derived As-bearing sulfur
+              | minerals deposited in the surface sediments releases As, which is temporarily
+              | immobilized through adsorption onto Fe(III) oxides, hydroxides, and oxyhydroxides
+              | (collectively referred to as oxides) in surrounding sediment matrix. Subsequent reductive
+              | dissolution of Fe(III) oxides and As(V) reduction under reducing conditions during the
+              | wet season leads to desorption and partitioning of As into the aqueous phase.
+              | Arsenic(V), the predominant oxidation state under oxic conditions, is generally
+blank         | 
+meta          | 96
+text          | considered the less mobile species; in contrast, As(III) dominates under reducing
+              | conditions and, although adsorbs more extensively onto Fe oxides, is loosely bound and
+              | thus more mobile under flow conditions. Therefore, characterization of factors and
+              | processes responsible for the reduction of As(V) is crucial toward understanding As
+              | transport.
+              |          Under anaerobic conditions, a major pathway contributing to the transformation
+              | of As(V) is microbial respiration of As(V), which has been shown to provide greater
+              | energetic yield than respiration on goethite and hematite under environmental conditions
+              | in Cambodian sediments (6). Dissimilatory As(V)-reducing bacteria (DARB) have been
+              | isolated from a wide range of environments and are phylogenetically and physiologically
+              | diverse (7). A number of studies have identified and characterized enzymes that catalyze
+              | As(V) respiration (8-11). The reductase is a periplasmic heterodimer composed of the
+              | terminal reductase, ArrA (87-110 kDa), and ArrB (25.7-34 kDa), which provides
+              | electrons to ArrA from c-type cytochromes (9-11).
+              |          Model organisms are invaluable for deciphering biochemical mechanisms
+              | responsible for As transformation under constrained laboratory conditions; however,
+              | these organisms and their functional genes may not be representative of those found in
+              | the environment. To this end, the diversity of arrA has only been explored in a limited
+              | number of environments, namely estuarine sediments of Chesapeake bay (12), aquifer
+              | sediments from West Bengal (13), and As-rich soda lakes (14, 15). Previously, Lear et
+              | al. (16) examined arrA genes in acetate-amended Cambodian sediments collected from 9
+              | m depth after 16 and 30 days of incubations, but were unable to amplify any products
+              | from unamended samples. Furthermore, only 10 arrA sequences were reported in that
+              | study, warranting further investigation of representative arrA genes within Cambodian
+              | sediments. Here, we characterize the diversity of As(V)-respiring bacteria in sediments
+              | from the Mekong Delta of Cambodia, where it has been shown that reduction-oxidation
+              | processes occurring within near-surface sediments (* 4 m below surface) are responsible
+              | for As release to the aquifer (2, 4). We report successful amplification and analysis of
+              | 223 arrA sequences (length $ 570 bp) from naturally occurring (unamended) surface
+              | sediments, increasing the existing sequence database by more than two-fold and
+meta          |                                                                                      97
+text          | significantly expanding the known diversity of this gene. Furthermore, we perform
+              | permutational multivariate analysis of variance (PERMANOVA; adonis function of the
+              | vegan package (17) in R (18)) on UniFrac distances to ascertain how variation in arrA
+              | sequences can be attributed to different environmental covariates (i.e., porewater
+              | constituents) within the soil profile. Overall, this analysis suggests that variation among
+              | arrA ecotypes at each site is best explained by porewater As and depth.
+blank         | 
+              | 
+title         | 5.3 Materials and Methods
+blank         | 
+title         | 5.3.1 Site description and soil sampling.
+text          |          Soil samples were obtained from four sites (Site A, B, C, and T) in Kien Svay
+              | district of Kandal province, Cambodia, between the Mekong and Basaac Rivers
+              | approximately 9 km southeast of capital city of Phnom Penh—a site representative of the
+              | Mekong floodplain system. Details of the biogeochemistry and geology of the site have
+              | been thoroughly discussed previously (2, 4, 19). Near surface intact cores were sampled
+              | using the procedure described in (4). Briefly, intact core samples were collected by hand-
+              | augering to 10, 50, 100, 200, and 400 cm depth with a soil sampling device fitted with a
+              | slide hammer (AMS) and core sampler containing a polycarbonate sleeve. The surface 1-
+              | 3 m of orange (Fe(III)-rich) sediment is overlain by gray clay, followed by gray sands at
+              | approximately 12 m depth. Intact cores were kept in gas-tight bags with Anaeropacks
+              | (Mitsubishi) to maintain anaerobic conditions.        Core samples were homogenized
+              | aseptically using mortar and pestal in an anaerobic chamber, and triplicate subcores were
+              | taken with sterile 5-mL syringes and sealed in anaerobic containers stored at -80 ˚C prior
+              | to DNA extraction.
+blank         | 
+title         | 5.3.2 Lysimeter installation and water collection.
+text          |          Tension lysimeters (Prenart Equipment ApS) were installed at the corresponding
+              | depths at each site prior to the collection of intact cores.     The procedure used for
+              | installing tension lysimeters was described in Kocar et al. (4). Holes were dug using a
+              | hand auger and sediments removed were used as back-fill after lysimeter installation.
+meta          | 98
+text          | Aqueous chemistry analysis. Lysimeter samples were immediately filtered through a 0.2
+              | µm membrane after collection. Total dissolved As was measured with hydride generation
+              | inductively coupled plasma atomic emission spectroscopy (HG-ICP-AES) with a
+              | detection limit of 5 µg L-1. Total dissolved Fe, Mn, Ca, Na, K, S, Mg, and P were
+              | measured by ICP-AES (Table 5.1). Samples were collected in triplicate and averaged
+              | values were used in multivariate analysis.
+blank         | 
+text          | Table 5.1 Concentration of aqueous constituents in porewater samples collected in Kien Svay,
+              | Kandal Province, Cambodia
+blank         | 
+text          |  #   Site     Depth   Season     As        Ca       Fe       K        Mg      Mn      Na        P       S
+              |                                (µg L-1)                               (mg L -1)
+              |  1    A         10     Dry       8.26     164.62    3.47     0.58       0     5.25    44.27      0      9.48
+              |  2    A         50     Dry      11.37     175.77    2.65     0.46    38.74     4.3    36.01    0.04     4.91
+              |  3    A        200     Dry     332.79       86.7   16.11     2.35    23.51    1.03    22.37    0.39     0.53
+              |  4    A        400     Dry     367.03     118.64   16.88     0.63    32.94    1.09    28.42    0.42     0.49
+              |  5    T         50     Dry      34.19     149.27    0.27     3.39    48.56    3.85   113.31    0.07     21.1
+              |  6    C        400     Dry     162.43      73.64    8.91     1.54    39.98    1.01    86.88    1.42    12.01
+              |  7    C         10     Dry       6.12      20.41    0.07     1.14     6.08     0.1     6.18      0      0.74
+              |  8    B        200     Wet      26.86       8.51    1.93     5.43    26.97    0.03    44.09    0.24   102.47
+              |  9    B        100     Wet        4.7      11.91    0.45   133.39    47.05    0.07    44.99    0.05    62.43
+              | 10    B         10     Wet       5.83       5.67    1.05    10.07    14.81     0.3    16.32    0.11     19.7
+              | 11    T        100     Wet       24.7     153.99     0.6    19.79    51.98    4.19    68.93    0.03     4.25
+              | 12    A        100     Wet       7.49        7.8    0.71    24.66    34.62    0.33    20.17    0.16     3.71
+              | 13    A        200     Wet     195.92     107.33   15.48     1.23    30.66    1.06    20.58    0.25     0.61
+              | 14    A         50     Wet      13.65      84.58    1.56    10.22    38.05    1.98    30.02    1.24    14.08
+              | 15    A         10     Wet       9.68      163.4    2.12     0.58       0     4.73    49.61      0     12.51
+blank         | 
+              | 
+              | 
+title         | 5.3.3 Genomic DNA extraction and PCR amplification of arsenate reductase
+              | (arrA) genes.
+text          |             DNA was extracted from soil (0.5 g) using FastDNA spin kit for soil (Qbiogene,
+              | Inc.) including use of FastPrep instrument to ensure cell lysis of Gram-positive bacteria,
+              | according to manufacturer’s instructions. Partial arrA gene sequences were amplified
+              | using a nested PCR approach designed by (12).                    Briefly,    primers As1F (5’-
+              | CGAAGTTCGTCCCGATHACNTGG-3’) and As1R (5’- GGGGTGCGGTCYTTNARY
+              | TC-3’) were used for PCR round 1 with a cycle of 5 min denaturation at 94 ˚C, followed
+              | by 35 cycles of 30 s denaturation at 94 ˚C, primer annealing of 30 s at 50 ˚C, followed by
+blank         | 
+meta          |                                                                                                99
+text          | 1 min extension at 72 ˚C. The nested PCR amplification was performed with As2F (5’-
+              | GTCCCNATBASNTGGGANRARGCNMT-3’) and As1R.                      PCR amplifications were
+              | performed in a total volume of 40 µL containing 20 µL 2X PCR Premix F (Epicentre),
+              | 0.5 µM of each primer, and 1.25 U AmpliTaq DNA polymerase (Applied Biosystems).
+              | 5.3.4 Cloning and sequencing PCR products.
+              |            Triplicate PCR products were pooled and gel purified using Qiaquick gel
+              | extraction kit (Qiagen) and purified product was cloned into vector pCR2.1 using TOPO-
+              | TA cloning kit (Invitrogen) following manufacturer’s instruction. Transformed cells
+              | were plated on agar plates containing 50 µg mL-1 kanamycin with X-gal. White colonies
+              | were transferred to 96-well plates containing LB broth with 50 µg mL-1 kanamycin,
+              | grown overnight at 37 ˚C and screened for PCR inserts using T7 and M13R vector
+              | primers.     Clones were sequenced on ABI 3730xl capillary sequencers (Applied
+              | Biosystems).
+blank         | 
+title         | 5.3.5 Quality control and phylogenetic analysis of arrA gene sequences.
+text          |            The raw arrA gene sequences were first trimmed of vector and primer sequence
+              | using the relevant tools in Geneious v5.4 (20) (~368 reads). All reads with trimmed-
+              | length greater than 570 bp were exported to FASTA format and processed/trimmed to the
+              | appropriate arrA open-reading frame using a custom script written with Biostings/R (21).
+              | Trimmed arrA sequences that were still larger than 570 bp and had unambiguous
+              | similarity with known arrA were translated and imported back into Geneious as amino
+              | acid sequences.
+              |            Reference amino acid sequences of ArrA were obtained by generally querying
+              | the public databases using PSI-BLAST (22) and several different ArrA sequences as
+              | query. We also took 190 amino acids as a minimum length requirement for any reference
+              | sequences from other studies. Using this approach, we retrieved and included arrA
+              | sequences derived from the studies of Song et al. (12) and Hery et al. (13), as well as
+              | sequences from cultured isolates, which include Bacillus selenitireducens MLS10
+              | (AAQ19491), Chrysiogenes arsenatis DSM 11915 (AAU11839), Sulfurospirillum
+              | barnesii SES-3 (AAU11840), Bacillus arseniciselenatis (AAU11841), Shewanella sp.
+meta          | 100
+text          | HAR-4 (AAU11842), Desulfosporosinus sp. Y5 (ABB02056), Bacillaceae bacterium
+              | ML-SRAO (ABW38092), Halarsenatibacter silvermanii (ACF74513), Wolinella
+              | succinogenes DSM 1740 (NP_906980), Geobacter uraniireducens Rf4 (YP_001229259),
+              | Alkaliphilus metalliredigens QYMF (YP_001318866), Alkaliphilus oremlandii OhILAs
+              | (YP_001512638), Natranaerobius thermophilus JW/NM-W N-LF (YP_001916826),
+              | Geobacter lovleyi (YP_001951391), Shewanella piezotolerans WP3 (YP_002311519),
+              | Desulfitobacterium hafniense DCB-2 (YP_002457721), Denitrovibrio acetiphilus DSM
+              | 12809 (YP_003504839), Bacillus selenitireducens MLS10 (YP_003700672), Ferrimonas
+              | balearica DSM 9799 (YP_003913347), Desulfurispirillum indicum S5 (YP_004111690),
+              | Desulfitobacterium hafniense Y51 (YP_520364), Shewanella sp. W3-18-1 (YP_964317),
+              | +-proteobacterium MLMS-1 (ZP_01288668), and Desulfonatronospira thiodismutans
+              | ASO3-1 (ZP_07017810).
+              |          Alignment of the arrA sequences from this study and the reference sequences
+              | was performed using MAFFT, and refined iteratively in Geneious. A total of 376
+              | sequences of at least 190 amino acids (including 223 novel sequences from the present
+              | study) remained in the final alignment after further quality trimming and the manual
+              | removal of any remaining aberrant/problematic sequences. A putative molybdopterin
+              | oxidoreductase from Burkholderiales bacterium 1_1_47 (ZP_07342827) was included as
+              | an outgroup. The final trimmed amino acid alignment (211 positions) was used as input
+              | for phylogenetic reconstruction using the maximum likelihood implementation in phyml
+              | (23), taking as optional parameters the Whelan and Goldman (24) substitution model and
+              | a discrete gamma model with two categories. The maximum likelihood tree was
+              | recalculated in 100 bootstrap iterations to assess node support.
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                   101
+text          | Table 5.2 Weighted UniFrac distance matrix used in PERMANOVA analysis. Definition of
+              | sample number with corresponding sample information is provided in Table 1.
+blank         | 
+text          |         1     2      3      4      5      6      7      8      9     10     11     12     13     14
+              |  2    0.62
+              |  3    0.61    0.6
+              |  4    0.56   0.29   0.58
+              |  5    0.57   0.66   0.62   0.62
+              |  6    0.76   0.72   0.78    0.6    0.8
+              |  7    0.58   0.56   0.58   0.56   0.52   0.71
+              |  8    0.61   0.57   0.64   0.46   0.67   0.49   0.57
+              |  9    0.55   0.59   0.59   0.48   0.54   0.59   0.43   0.37
+              | 10    0.54   0.57   0.54   0.54   0.47   0.76   0.34   0.62   0.46
+              | 11    0.58   0.55   0.57   0.57   0.53   0.72    0.3   0.57   0.46   0.22
+              | 12    0.63   0.55   0.63   0.53   0.58   0.71   0.62   0.57    0.6   0.64   0.63
+              | 13    0.59   0.64   0.65    0.6   0.39    0.8   0.64   0.66   0.65   0.63   0.66   0.56
+              | 14    0.67   0.45   0.65   0.45   0.72   0.62   0.57   0.47    0.6   0.65   0.61   0.53   0.69
+              | 15    0.59   0.65   0.65   0.62   0.37   0.81   0.62   0.68   0.63    0.6   0.63   0.58   0.12   0.71
+blank         | 
+title         | 5.3.6 Multivariate analysis of arrA genes and environmental factors.
+text          |          In order to assess differences in the arrA ecotypes present in a particular sample,
+              | we calculated the weighted-UniFrac distance metric for all pair-wise combinations of
+              | samples (Table 5.2). The weighted-UniFrac calculation requires a species-abundance
+              | table, which by implication requires a species definition for arrA. We took a somewhat
+              | strict 98% amino acid similarity cutoff (as measured in branch-length on the phylogenetic
+              | tree) as the species definition to build the abundance table, using the tipglom function
+              | in the phyloseq package.          The subsequent arrA species abundance matrix and
+              | phylogenetic tree were provided as input to weighted-UniFrac, as implemented in
+              | phyloseq/R. The resulting weighted-UniFrac distance matrix was used in PERMANOVA
+              | analysis using the adonis function in the vegan package of R.
+blank         | 
+              | 
+title         | 5.4 Results and Discussion
+blank         | 
+title         | 5.4.1 Phylogenetic analysis of arrA genes in Cambodian sediments.
+text          |          The diversity of As(V)-reducing bacteria was assessed in near-surface
+              | Cambodian sediments by amplifying the arrA functional gene from sediment samples
+meta          | 102
+text          | without prior enrichment (e.g., organic carbon amendment). A total of 376 non-chimeric
+              | sequences were included in a maximum likelihood estimate of the ArrA phylogeny
+              | (Figure 5.1).   We arbitrarily defined 0.02 average amino acid substitutions per site
+              | (branch length) as a cutoff for a stringent definition of an operational taxonomic unit
+              | (OTU), calculated by the tipglom function in phyloseq, resulting in a total of 173 ArrA
+              | phylotypes. In general, putative ArrA sequences obtained from Cambodia sediment
+              | formed a separate monocladistic group that is most closely related to the arrA gene in
+              | Geobacter uranireducens and Geobacter lovleyii (Figure 1).
+              |          Sequences acquired in the present study were compared phylogenetically to
+              | environmental predicted ArrA sequences from Chesapeake Bay (12) and West Bengal
+              | (13), made possible by the application of the same nested PCR method and primer set in
+              | all three studies, which provided highly comparable product length and arrA region
+              | amplified. A great majority (>90%) of sequences acquired from near-surface Cambodian
+              | sediments form a distinct cluster from the Chesapeake Bay and West Bengal ArrA
+              | sequences. The remaining sequences fell within the DARB isolate cluster, with closest
+              | neighbor sequences from Chesapeake Bay. Previously, sequences from Chesapeake Bay
+              | were reported to form distinct phylogenetic clusters from DARB isolates; however,
+              | application of maximum likelihood rather than neighbor-joining method for tree
+              | construction placed 51 (70%) sequences from Chesapeake Bay within the DARB isolate
+              | cluster. Although neighbor-joining method is computationally efficient, it has several
+              | well-understood weaknesses that make it a poor choice for estimating complex
+              | phylogenies, whereas estimating phylogeny using maximum likelihood is demonstrably
+              | more robust and consistent when encountering assumption violations (25). The ArrA
+              | phylogenetic trees presented here are the first to be constructed with maximum likelihood
+              | method and may provide a more accurate estimate of the relationship between clones
+              | from various sites.   Hery et al. (13) states that 10 arrA sequences acquired from
+              | Cambodian sediments after enrichment/stimulation with As(V) and acetate amendments
+              | (16) cluster separately from West Bengal sequences; however, sequences from that study
+              | are unavailable and thus we are unable to make comparisons to those obtained within our
+              | current study. Nevertheless, our current dataset supports the conclusion that clones from
+meta          |                                                                                      103
+text          | Figure 5.1 Maximum likelihood phylogram of available ArrA sequences. Sequences were
+              | required to be at least 190 amino acids in length for inclusion in analysis. Sequences obtained
+              | from two previous environmental surveys (Song et al. (12); Hery et al. (13)) are also included.
+              | Tips (phylotypes) are labeled according to their source site and depth, the previous survey, or
+              | with an isolate name, respectively. Multiple symbols at the same tip indicate that the same
+              | phylotype was observed in multiple samples. The number inside the symbol indicates the total
+              | number of individuals observed.
+blank         | 
+              | 
+              | 
+meta          | 104
+text          | Figure 5.2 Expanded representation of the Cambodia-specific clade (this study).
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                   105
+text          | Table 5.3 Permutational MANOVA results using adonis function of vegan package.
+blank         | 
+text          |                                Sums of       Mean
+              |  Variable         Df                                    F Model        R2         P
+              |                                Squares      Squares
+              |   Depth           1             0.29          0.29        2.15        0.12       0.06
+              |    Site           3             0.57          0.19        1.41        0.23       0.22
+              |    As             1             0.28          0.28        2.12        0.12       0.07
+              |    Ca             1             0.15          0.15        1.14        0.06       0.38
+              |    Fe             1             0.15          0.15        1.13        0.06       0.40
+              |     K             1             0.05          0.05        0.35        0.02       0.96
+              |    Mg             1             0.20          0.20        1.47        0.08       0.21
+              |    Mn             1             0.17          0.17        1.30        0.07       0.30
+              |    Na             1             0.24          0.24        1.75        0.10       0.12
+              |     P             1             0.10          0.10        0.74        0.04       0.71
+              |     S             1             0.12          0.12        0.90        0.05       0.55
+              | Cambodia are indeed distinct from those amplified from West Bengal. A more detailed
+              | view of the phylogeny of arrA clones from Cambodia is provided in Figure 5.2. Clones
+              | do not appear to form obvious clusters by site, depth, or season (wet-season samples are
+              | indicated by black outline).
+blank         | 
+title         | 5.4.2 Permutational MANOVA analysis.
+text          |           To determine whether variations in environmental factors can explain variations
+              | observed in arrA sequence data, we applied permutational multivariate analysis of
+              | variance (PERMANOVA) using distance matrix calculated by weighted UniFrac method
+              | (26). PERMANOVA is a non-parametric method for multivariate analysis of variance
+              | that is less stringent than traditional multivariate analyses and is, therefore, more
+              | appropriate for ecological data (27). We were able to determine whether variation in
+              | porewater chemistry (provided in Table 5.1) explained variations in As(V)-reducing
+              | microbial community structure.           Results indicate that variation in depth and As
+              | concentrations in porewater are weakly associated (P = 0.06 and 0.07, respectively) with
+              | variations in arrA sequences between samples, while other pore-water aqueous
+              | constituents do not appear significant (Table 5.3).
+              |           Variations in As(V)-reducing microbial communities associated with depth and
+              | As concentrations can be explained by shifts in active redox processes with increasing
+blank         | 
+meta          | 106
+text          | depth within the surface soils. Dry season conditions lead to the formation of large
+              | cracks, due to the great shrink-swell capacity of clays, leading to aeration and adsorption
+              | of As on Fe(III) oxides at shallow depths (< 1 m) (4). However, deeper depths (>1 cm)
+              | are highly reducing throughout both the dry and wet seasons, where sediment color
+              | transitions to gray and As concentrations are no longer strongly correlated with Fe and
+              | alkalinity (4). Arsenic(V)-respiring microorganisms are most likely active in the near
+              | surface sediments where fresh carbon sources are deposited annually (e.g. detritus from
+              | plants and animals) and As(V) is available. At depths >1 m, As is present mostly in the
+              | reduced form and, therefore, As(V)-respiration likely plays a minor role in As cycling.
+blank         | 
+              | 
+title         | 5.5 References
+ref           | 1.     Nickson R, McArthur J, Ravenscroft P, Burgess W, & Ahmed K (2000)
+              |        Mechanism of arsenic release to groundwater, Bangladesh and West Bengal.
+              |        Appl Geochem 15(4):403-413 .
+              | 2.     Polizzotto ML, Kocar BD, Benner SG, Sampson M, & Fendorf S (2008) Near-
+              |        surface wetland sediments as a source of arsenic release to ground water in Asia.
+              |        Nature 454 (7203):505-U505 .
+              | 3.     Berg M, et al. (2001) Arsenic contamination of groundwater and drinking water
+              |        in Vietnam: a human health threat. Environ Sci Technol 35(13):2621-2626 .
+              | 4.     Kocar BD, et al. (2008) Integrated biogeochemical and hydrologic processes
+              |        driving arsenic release from shallow sediments to groundwaters of the Mekong
+              |        delta. Appl Geochem 23(11):3059-3071 .
+              | 5.     Buschmann J, Berg M, Stengel C, & Sampson ML (2007) Arsenic and manganese
+              |        contamination of drinking water resources in Cambodia: Coincidence of risk areas
+              |        with low relief topography. Environmental Science & Technology 41(7):2146-
+              |        2152 .
+              | 6.     Kocar BD & Fendorf S (2009) Thermodynamic Constraints on Reductive
+              |        Reactions Influencing the Biogeochemistry of Arsenic in Soils and Sediments.
+              |        Environ Sci Technol 43(13):4871-4877 .
+              | 7.     Oremland R, et al. (2005) A microbial arsenic cycle in a salt-saturated, extreme
+              |        environment. Science 308(5726):1305-1308 .
+              | 8.     Saltikov C, Cifuentes A, Venkateswaran K, & Newman D (2003) The ars
+              |        detoxification system is advantageous but not required for As(V) respiration by
+              |        the genetically tractable Shewanella species strain ANA-3. Appl Environ Microb
+              |        69(5):2800-2809 .
+              | 9.     Saltikov C & Newman D (2003) Genetic identification of a respiratory arsenate
+              |        reductase. P Natl Acad Sci USA 100(19):10983-10988 .
+blank         | 
+              | 
+meta          |                                                                                        107
+ref           | 10.   Krafft T (1998) Purification and characterization of the respiratory arsenate
+              |       reductase of Chrysiogenes arsenatis - Krafft - 2001 - European Journal of
+              |       Biochemistry - Wiley Online Library. Eur. J. Biochem.
+              | 11.   Afkar E, et al. (2003) The respiratory arsenate reductase from Bacillus
+              |       selenitireducens strain MLS10. Fems Microbiol Lett 226(1):107-112 .
+              | 12.   Song B, Chyun E, Jaffe PR, & Ward BB (2009) Molecular methods to detect and
+              |       monitor dissimilatory arsenate-respiring bacteria (DARB) in sediments. Fems
+              |       Microbiol Ecol 68(1):108-117 .
+              | 13.   Hery M, et al. (2010) Arsenic release and attenuation in low organic carbon
+              |       aquifer sediments from West Bengal. Geobiology 8(2):155-168 .
+              | 14.   Hoeft SE, Kulp TR, Han S, Lanoil B, & Oremland RS (2010) Coupled
+              |       Arsenotrophy in a Hot Spring Photosynthetic Biofilm at Mono Lake, California.
+              |       Appl. Environ. Microbiol. 76(14):4633.
+              | 15.   Kulp TR, et al. (2006) Dissimilatory arsenate and sulfate reduction in sediments
+              |       of two hypersaline, arsenic-rich soda lakes: Mono and Searles lakes, California.
+              |       Appl. Environ. Microbiol. 72(10):6514-6526 .
+              | 16.   Lear G, Song B, Gault AG, Polya DA, & Lloyd JR (2007) Molecular analysis of
+              |       arsenate-reducing bacteria within Cambodian sediments following amendment
+              |       with acetate. Appl Environ Microb 73(4):1041-1048 .
+              | 17.   Jari Oksanen FGB, Roeland Kindt, Pierre Legendre, R. B. O'Hara GLS, Peter
+              |       Solymos, M. Henry H. Stevens, & Wagner aH (2011) Vegan: Community
+              |       Ecology Package), R package version 1.17-11.
+              | 18.   R development core team RDC (2011) R: A language and environment for
+              |       statistical computing. R Foundation for Statistical Computing.
+              | 19.   Benner SG, et al. (2008) Groundwater flow in an arsenic-contaminated aquifer,
+              |       Mekong Delta, Cambodia. Appl Geochem 23(11):3072-3087 .
+              | 20.   Drummond AJ KM, Heled J, Moir R, Thierer T, Ashton B (2006) Geneious
+              |       v1.2.1), Available from http://www.geneious.com/.
+              | 21.   DebRoy HPaPAaRGaaS (2009) Biostrings: String objects representing biological
+              |       sequences, and matching algorithms), R package version 2.20.0.
+              | 22.   Altschul S (1997) Gapped BLAST and PSI-BLAST: a new generation of protein
+              |       database search programs. Nucleic Acids Res. 25(17):3389-3402.
+              | 23.   Guindon S, et al. (2010) New Algorithms and Methods to Estimate Maximum-
+              |       Likelihood Phylogenies: Assessing the Performance of PhyML 3.0. Syst. Biol.
+              |       59(3):307-321 .
+              | 24.   Whelan S (2001) A General Empirical Model of Protein Evolution Derived from
+              |       Multiple Protein Families Using a Maximum-Likelihood Approach. Mol. Biol.
+              |       Evol.
+              | 25.   Huelsenbeck JP (1995) The robustness of two phylogenetic methods: four-taxon
+              |       simulations reveal a slight superiority of maximum likelihood over neighbor
+              |       joining. Mol Biol Evol 12(5):843-849 .
+              | 26.   Lozupone CA, Hamady M, Kelley ST, & Knight R (2007) Quantitative and
+              |       qualitative beta diversity measures lead to different insights into factors that
+              |       structure microbial communities. Appl. Environ. Microbiol. 73(5):1576-1585 .
+blank         | 
+meta          | 108
+ref           | 27.   Anderson M (2001) A new method for non"parametric multivariate analysis of
+              |       variance. Austral Ecol.
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                             109
+              | 110
+title         | Chapter 6: Summary
+blank         | 
+text          |          The fate and transport of As in soils and sediments is determined by a complex
+              | interplay of chemical, biological, and physical factors. To this end, this thesis aimed to
+              | decipher the combined effects of biotic and abiotic reactions controlling As redox,
+              | sorption, and transport processes within systems that simulate the physical complexity of
+              | soil systems.
+              |          Mass transfer limitations within soil systems causes steep concentration
+              | gradients that form redox interfaces. Competitive reduction and oxidation reactions may
+              | take place at such boundaries, altering the fate of As due to fluctuations in As speciation.
+              | In Chapter 2, I show that at the interface of a strong oxidant, birnessite, and reductant,
+              | As(V)-reducing bacteria, As can be cycled between oxidized and reduced forms.
+              | Arsenic(III) is rapidly oxidized by birnessite, producing As(V) which can then be
+              | reduced back to As(III).     The oxidation of As(III) reductively dissolves birnessite,
+              | releasing Mn(II) into solution. During dissimilatory As(V) reduction, lactate oxidation
+              | also results in the production of (bi)-carbonate. Initially, I hypothesized that the final
+              | ratio of As(V) to As(III) would be determined by the ratio of birnessite relative to the
+              | electron donor used for microbial respiration. Theoretically, As(V) would continue to be
+              | generated until the exhaustion of the oxidant (birnessite) and, conversely, As(III)
+              | concentrations would be expected to diminish only after lactate source is depleted.
+              | However, I discovered that the redox cycle becomes short-circuited in the presence of
+              | chemically and biologically produced reaction products: the products of the As oxidation
+              | and reduction reactions combine to precipitate rhodochrosite (MnCO3), which
+              | subsequently inhibits As(III) oxidation through birnessite surface passivation. Reactive
+              | transport modeling predictions demonstrated that decreased supply of lactate decreases
+              | the extent of As(III) production due to limiting carbon source for As(V) respiration,
+              | which in turn decreases rhodochrosite formation and promotes As(III) oxidation by
+              | birnessite. It was also observed that pseudo-steady state is reached within reducing and
+blank         | 
+meta          |                                                                                         111
+text          | oxidizing environments due to the faster relative rate of redox processes as compared to
+              | diffusion limited mass transport of solutes. This finding has important implications on
+              | the speciation of As in soils where physical complexity can induce separated
+              | biogeochemical islands that interact through diffusion controlled mass transfer. My
+              | results demonstrate that products from biologically driven reaction products can alter the
+              | expected rates and extent of redox processes taking place at redox interfaces within
+              | diffusion limited systems like soils.
+              |          Although Chapter 2 explored the role of Mn oxides as an oxidant of As(III) that
+              | is reductively dissolved during the oxidation process, Mn oxides can be regenerated by
+              | Mn(II) oxidation pathways in soil systems, controlling the concentration of Mn(II) in
+              | solution. A majority of Mn oxides in the environment are biogenically produced (1),
+              | where the oxidative and sorptive properties of biogenic manganese oxides differ from
+              | those of synthetic Mn oxides. The presence of Mn(II)-oxidizing bacteria may maintain a
+              | lower aqueous concentration of Mn(II) through precipitation of Mn oxides, preventing
+              | the formation of rhodochrosite, and thus allowing rapid redox cycling of As to be
+              | maintained. Further exploration is needed to determine the role of biological processes in
+              | Mn oxidation and its influence on As redox cycling.
+              |          The combined effects of oxidation and competitive adsorption of As onto
+              | multiple sorbents available in soils can greatly impact the release of As from the solid
+              | phase. In Chapter 3, I demonstrate that in the presence of both birnessite and goethite,
+              | As(III) is rapidly oxidized to As(V) by birnessite, and oxidation rate is dependent upon
+              | birnessite mass and As(III) concentrations, consistent with previous findings (2, 3).
+              | Arsenic(V)    then   preferentially     partitions   onto   goethite   with   aqueous   As(V)
+              | concentrations determined by the degree of surface coverage; saturation of the goethite
+              | surface leads to a proportional increase in the aqueous concentration. My findings show
+              | that within soil matrices composed of a mixture of Mn and Fe oxides, Mn oxides operate
+              | primarily as oxidants of As(III) to form the more strongly sorbing As(V); ultimately, Fe
+              | oxides are the primary adsorbents immobilizing As(V) within soil systems.
+              |          I examine the combined effects of competitive reduction-oxidation reactions
+              | studied in Chapter 2 with the competitive sorption processes explored in Chapter 3within
+meta          | 112
+text          | a system simulating the conditions imposed by the complex structure of soils.
+              | Accordingly, in Chapter 4, I reveal the mechanisms involved in As cycling within
+              | synthetic aggregates composed of Mn and Fe oxide coated sands inoculated with As(V)-
+              | reducing bacteria.    The release and speciation of dissolved As, Mn, and Fe from
+              | aggregates is highly dependent upon redox status.        Even as the advecting solution
+              | surrounding the aggregate is aerated, a reducing interior is formed where As, Mn, and Fe
+              | are desorbed and mobilized to the oxygenated exterior realm of the aggregate. Reduced
+              | species of As, Mn, and Fe are transported from the aggregate center and accumulate on
+              | the exterior rind of the aggregate. Upon transition to anaerobic conditions in advecting
+              | solution, a pulse of all three constituents is released into the advecting solution.
+              | Conversely, transition from anaerobic to aerated conditions leads to decrease in release of
+              | reduced species into the advecting channel because of oxidation precipitation of Fe and
+              | Mn oxides. Additionally, As(III) oxidation by Mn oxides is only appreciable in the
+              | presence of O2; the rapid dissimilatory reduction of Mn oxides after the onset of anoxic
+              | conditions inhibits further oxidation. Collectively, these results show that As retention is
+              | dependent upon redox conditions within soil, where retention and release can be highly
+              | variable, spatially and temporally.
+              |          In addition to chemical and physical heterogeneity, biological diversity
+              | contributes to the complexity of operative processes within soil systems. Accordingly, I
+              | surveyed the microbial ecology of As(V) reduction in soils of the Mekong Delta, where
+              | As cycling plays a crucial role in the health and safety of millions. Dissimilatory As(V)-
+              | respiring bacteria (DARBs) have been shown to catalyze the reduction of As in many
+              | environments (5). However, the community structure of DARBs, which can reflect local
+              | chemical conditions, has not been thoroughly explored in the environment. In Chapter 5,
+              | I discovered that sequences encoding functional gene arrA amplified from the Mekong
+              | Delta of Cambodia composed a novel, phylogenetically distinct clade. Furthermore,
+              | application of permutational multivariate analysis of variance using distance matrix
+              | calculated from the constructed phylogenetic tree demonstrates that variations in
+              | community structure can be explained by variations in porewater As concentration and
+              | soil depth. However, changes in community structure of As(V)-reducing bacteria is
+meta          |                                                                                         113
+text          | influenced by a multitude of factors aside from aqueous concentrations of select
+              | constituents; therefore, additional sequence information along with corresponding
+              | environmental chemical measurements is needed to confirm these findings.
+blank         | 
+              | 
+              | 
+              | 
+meta          | 114
+title         | 6.1 References
+blank         | 
+ref           | 1.   Diem D & Stumm W (1984) ScienceDirect - Geochimica et Cosmochimica Acta :
+              |      Is dissolved Mn2+ being oxidized by O2 in absence of Mn-bacteria or surface
+              |      catalysts? Geochim. Cosmochim. Acta.
+              | 2.   Manning B, Fendorf S, Bostick B, & Suarez D (2002) Arsenic(III) oxidation and
+              |      arsenic(V) adsorption reactions on synthetic birnessite. Environ Sci Technol
+              |      36(5):976-981 .
+              | 3.   Scott M & Morgan J (1995) Reactions at oxide surfaces. 1. Oxidation of As(III)
+              |      by synthetic birnessite.Environ Sci Technol 29(8):1898-1905.
+              | 4.   Masue-Slowey Y, Kocar BD, Jofre SAB, Mayer KU, & Fendorf S (2011)
+              |      Transport Implications Resulting from Internal Redistribution of Arsenic and Iron
+              |      within Constructed Soil Aggregates. Environ. Sci. Technol. 45(2):582-588.
+              | 5.   Oremland R & Stolz J (2003) The ecology of arsenic. Science 300(5621):939-
+              |      944 .
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                   115
+              | 116
+              | APPENDIX A: Supporting information for
+              | Chapter 2
+blank         | 
+title         | A.1 Reactive Transport Modeling using MIN3P
+blank         | 
+text          |         The framework of our simulation is divided into a finite difference matrix
+              | consisting of three cells representing two chambers separated by a permeable membrane
+              | (representing the third cell). Dirichlet (fixed head) boundaries are set at both ends of the
+              | three-cell domain to simulate a closed system. The heads within both chambers remain
+              | identical at all times; therefore, there is no hydraulic gradient. Hence, hydraulic
+              | conductivities are not applicable in this system, and transport is solely governed by
+              | diffusion.
+              |         The biogeochemical conceptual model of our system is summarized in Table 2.1
+              | in Chapter 2, and can be divided into the following processes: 1) abiotic oxidation of
+              | As(III) by birnessite (eq. 1-3), 2) reductive dissolution of birnessite (eq. 4), 3) microbial
+              | reduction of As(V) with lactate oxidation to acetate (eq. 5), 4) adsorption/desorption of
+              | As(V) and As(III) from birnessite (eq. 6-8), and 5) precipitation of rhodochrosite (eq. 9).
+              |         Rhodochrosite precipitation is allowed upon Mn2+/CO32- reaching saturation, the
+              | formation of which inhibits As(III) oxidation by birnessite (eq. 1) by defining
+              | rhodochrosite species as an inhibition term with an inhibition constant (Ki) of 1.5x10-10.
+              | Arsenic adsorption/desorption is described using a simple reversible equilibrium
+              | expression formulated by Dzombeck and Morel (38) as described by Mayer et al. (36,
+              | 39). The diffusion coefficient was calibrated to As concentrations measured in chambers
+              | over time when As was injected in the absence of bacteria and birnessite.
+              |         Within the chamber cells, rates of inter-aqueous and heterogeneous reactions were
+              | described according to the general formulation:
+blank         | 
+              | 
+              | 
+              | 
+text          |               Where Vmax is the maximum rate of reaction, term (1) represents the rate
+              | dependence on substrate concentration [A] (multiple substrates are allowed), with
+              | exponent X providing reaction order (or fractional dependence), term (2) is a Monod
+              | expression with exponential dependence Y, where Sm represents a limiting substrate
+              | concentration and Km is a half-saturation constant, term (3) is an inhibition term with
+              | exponential dependence Z, where Ki represents an inhibition threshold concentration for
+blank         | 
+meta          |                                                                                          117
+text          |     substrate Si, and term (4) is a chemical affinity term, where IAP and Keq represent the ion
+              |     activity product and reaction equilibrium (or solubility product) constants, respectively.
+              |                     There is a paucity of adsorption isotherm data available for As on
+              |     birnessite; therefore, diffuse layer constants for As(V) adsorption on hydrous ferric
+              |     oxides (HFO) were applied in the model (using values from (1) shown in Table 2.1 of the
+              |     Chapter 2, Eqs 6 - 8).
+blank         | 
+              | 
+text          |     Table A.1 Rate expressions for reactions used in simulation.
+blank         | 
+text          | Eqn       (Bio)geoch-       Rate Expression
+              | No.       emical reaction
+              | 1         As(V)                                                      #            [C 3H 5O -3 ]             & y1 #        [As(V)]               &y2
+              |           Reduction         R(As(V)-reduction) = "k(As(V)-reduction)%% halfsat -[C H O - ]                  ( % halfsat -[As(V)]                (
+              |                                                                                                         - (
+              |                                                                      $ [K(As(V)-reduction) ] + [C 3H 5O 3 ] ' $ [K(As(V)-reduction) ] + [As(V)] '
+              |                                                                                    3 5 3
+blank         | 
+              | 
+              | 
+text          | 2         As(III)                                                       #            [MnO 2 ]             & y1 #         [As(III)]            &y2 #          thresh -[MnCO 3 ]
+              |                                                                                                                                                           K(As(III)-oxidation)     &z
+              |           Oxidation         R(As(III)-oxidation) = "k(As(III)-oxidation)% halfsat -[MnO 2 ]               ( % halfsat -[As(III)]              ( % thresh -[MnCO3 ]                 (
+              |                                                                         $ K(As(III)-oxidation) + [MnO 2 ] ' $ K(As(III)-oxidation) + [As(III)]' $ K(As(III)-oxidation) + [MnCO 3 ] '
+              |                   !
+              | 3         Rhodochrosite                                        #          [Mn 2+ ]             &y1 #             [CO 3 2- ]               &y 2
+              |           Precipitation     R(MnCO3 -pptn)   = "k(MnCO3 -pptn)%% halfsat -[Mn 2+ ]             ( %                                        (
+              |                     !                                                                      2+ ( %       halfsat -[CO 3 2- ]           2- (
+              |                                                                  [K
+              |                                                                $ (MnCO3 -pptn)     ] + [Mn   ]       [K
+              |                                                                                                ' $ (MnCO3 -pptn)            ] + [CO 3   ] '
+blank         | 
+text          |     * Kinetic and thermodynamic parameters given in table A.2. All exponential values (yn
+              |     and z) are set
+              |                 ! to 1.
+blank         | 
+              | 
+              | 
+              | 
+meta          |     118
+text          |    Table A.2 Kinetic and thermodynamic values used in reactive transport simulations.
+blank         | 
+              | 
+text          |                                                                                 Rhodochrosite
+              |                     As(V) reduction              As(III) oxidation
+              |                                                                                  precipitation
+              |     Rate
+              |   constant
+              | (mol L-1 d-1)b                1.0x10-8                       2.0x10-4a                1.0x10-9a
+blank         | 
+              | 
+text          |  Literature                   2.31x10-6
+              |                     H            (40)              --           ---                      ---
+              |   Values
+              |                                         -9
+              |                               2.90x10
+              |                      L           (27)              --           ---                      ---
+blank         | 
+              | 
+text          |   Half-
+              | Saturation
+              |  Constant
+              |    (Ks)
+blank         | 
+text          |                  C 3H 5O 3-   5.0x10-4a        Birnessite    2.0x10-4a    Mn2+        1.0x10-10a
+              |                   As(V)       1.0x10-10a        As(III)      5.0x10-4a    CO32-       1.0x10-10a
+blank         | 
+text          |  Inhibition
+              |   Constant
+              |     (Ki)
+blank         | 
+text          |                     --            --         Rhodochrosite   1.5x10-10a    --             --
+blank         | 
+              | 
+text          |    a = calibrated
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                                    119
+title         |     A.2 Carbonate calculations
+blank         | 
+text          |     Concentrations of carbonate were calculated according to the following reaction:
+blank         | 
+text          |     2H3AsO4 + C3H5O3- --> 2H3AsO3 + CO32- + H+ + CH3COOH
+blank         | 
+text          |     For every 1 mol of lactate oxidized via reduction of arsenate, 1 mol of carbonate and 1
+              |     mol of acetate are produced. pH of the solutions remained constant owing to the addition
+              |     of high concentration of PIPES buffer; therefore, the concentration of carbonate within
+              |     the system was equivalent to the concentration of acetate measured. The carbonate
+              |     concentrations calculated from these measurements were then used to calculate the
+              |     saturation index of rhodochrosite.
+blank         | 
+              | 
+              | 
+title         |     A.3 Saturation index calculations
+blank         | 
+text          |     The reaction representing the formation of rhodochrosite in the RTM is as follows:
+blank         | 
+text          |     Mn2+ + CO32- = MnCO3(s)
+blank         | 
+text          |     Saturation index of rhodochrosite is calculated as
+blank         | 
+              | 
+              | 
+              | 
+text          |     where
+blank         | 
+text          |     IAP = (Mn 2+ )(CO32" ) = # Mn 2+ [Mn 2+ ]# CO 2" [CO32" ]
+              |                                                   3
+blank         | 
+              | 
+              | 
+              | 
+text          |     calculated at pH 7.1.
+              | !   Ionic strength of the solution was low (0.018), therefore the Debye-Hückel equation was
+              |     used to calculate activity coefficients.
+blank         | 
+text          |     Rhodochrosite solubility product values (log [Ksp]) in literature range from -10.2 (41) to -
+              |     10.93(42). The intermediate value of -10.41 (43), was applied in this study.
+blank         | 
+              | 
+              | 
+              | 
+meta          |     120
+title         | A.4 Diffusion coefficient calculations
+blank         | 
+text          | The diffusion coefficient (DAB) use in the reactive transport model was calculated using
+              | the following formula:
+blank         | 
+              | 
+              | 
+text          | Where S [m2] is the surface area of the membrane, DAB [m2 s-1] is the diffusion coefficient
+              | for the solute, H [m3 g-1]is the partition coefficient, V [m3] is the volume of each
+              | compartment, and L [m] is the length of the reactor. " [mol L-1 s-1]is the slope of the line
+              | for a plot of y versus time, where
+blank         | 
+              | 
+              | 
+text          | Conc1 [mol L-1] is As concentration in the chamber where As was injected and Conc2
+              | [mol L-1]is the As concentration into which As is being diffused (here, an injection in
+              | only one chamber was conducted while in all other experiments dual injection
+              | transpired). An equal and constant head was applied to both chambers, creating a
+              | diffusion controlled transport system.
+blank         | 
+              | 
+              | 
+              | 
+title         | A.5 References cited in Appendix A
+blank         | 
+ref           | 1.     Dixit S & Hering J (2003) Comparison of arsenic(V) and arsenic(III) sorption
+              |        onto iron oxide minerals: Implications for arsenic mobility. Environ Sci Technol
+              |        37(18):4182-4189 .
+              | 2.     Manning B, Fendorf S, Bostick B, & Suarez D (2002) Arsenic(III) oxidation and
+              |        arsenic(V) adsorption reactions on synthetic birnessite. Environ Sci Technol
+              |        36(5):976-981 .
+              | 3.     Gupta SK & Chen KY (1978) Arsenic Removal by Adsorption. Journal Water
+              |        Pollution Control Federation 50(3):493-506 .
+              | 4.     Tufano KJ, Reyes C, Saltikov CW, & Fendorf S (2008) Reductive Processes
+              |        Controlling Arsenic Retention: Revealing the Relative Importance of Iron and
+              |        Arsenic Reduction. Environ Sci Technol 42(22):8283-8289 .
+              | 5.     Zobrist J, Dowdle P, Davis J, & Oremland R (2000) Mobilization of arsenite by
+              |        dissimilatory reduction of adsorbed arsenate. Environ Sci Technol 34(22):4747-
+              |        4753 .
+              | 6.     Myers C & Nealson K (1988) Bacterial manganese reduction and growth with
+              |        manganese oxide as the sole electron-acceptor. Science 240(4857):1319-1321 .
+blank         | 
+              | 
+meta          |                                                                                         121
+ref           | 7.    Mukhopadhyay R, Rosen B, Pung L, & Silver S (2002) Microbial arsenic: from
+              |       geocycles to genes and enzymes. Fems Microbiol Rev 26(3):311-325 .
+              | 8.    Oremland R & Stolz J (2003) The Ecology of Arsenic. Science 300(5621):939.
+              | 9.    Eary L & Shramke J (1990) Chemical Modeling of Aqueous Systems II (American
+              |       Chemical Society, Washington, DC).
+              | 10.   Tallman D & Shaikh A (1980) Redox stability of inorganic arsenic(III) and
+              |       arsenic(V) in aqueous-solution. Anal Chem 52(1):197-199 .
+              | 11.   Gihring T, Druschel G, McCleskey R, Hamers R, & Banfield J (2001) Rapid
+              |       arsenite oxidation by Thermus aquaticus and Thermus thermophilus: Field and
+              |       laboratory investigations. Environ Sci Technol 35(19):3857-3862 .
+              | 12.   Santini J, Sly L, Schnagl R, & Macy J (2000) A new chemolithoautotrophic
+              |       arsenite-oxidizing bacterium isolated from a gold mine: Phylogenetic,
+              |       physiological, and preliminary biochemical studies. Appl Environ Microb
+              |       66(1):92-97 .
+              | 13.   Oscarson D, Huang P, Defosse C, & Herbillon A (1981) Oxidative power of
+              |       Mn(IV) and Fe(III) oxides with respect to As(III) in terrestrial and aquatic
+              |       environments. Nature 291(5810):50-51 .
+              | 14.   Scott M & Morgan J (1995) Reactions at oxide surfaces. 1. Oxidation of As(III)
+              |       by synthetic birnessite. Environ Sci Technol 29(8):1898-1905 .
+              | 15.   Nealson K & Saffarini D (1994) Iron and manganese in anaerobic respiration -
+              |       environmental significance, physiology, and regulation. Annu Rev Microbiol
+              |       48:311-343 .
+              | 16.   Manning B, Fendorf S, & Goldberg S (1998) Surface structures and stability of
+              |       arsenic(III) on goethite: Spectroscopic evidence for inner-sphere complexes.
+              |       Environ Sci Technol 32(16):2383-2388 .
+              | 17.   Deschamps E, Ciminelli V, Weidler P, & Ramos A (2003) Arsenic sorption onto
+              |       soils enriched in Mn and Fe minerals. Clay Clay Miner 51(2):197-204 .
+              | 18.   Amirbahman A, Kent D, Curtis G, & Davis J (2006) Kinetics of sorption and
+              |       abiotic oxidation of arsenic(III) by aquifer materials. Geochim Cosmochim Ac
+              |       70(3):533-547 .
+              | 19.   Myrold D & Tiedje J (1985) Diffusional constraints on denitrification in soil. Soil
+              |       Sci Soc Am J 49(3):651-657 .
+              | 20.   Tokunaga T, et al. (2003) Distribution of chromium contamination and microbial
+              |       activity in soil aggregates. J Environ Qual 32(2):541-549 .
+              | 21.   Sexstone A, Revsbech N, Parkin T, & Tiedje J (1985) Direct measurement of
+              |       oxygen profiles and denitrificaiotn rates in soil aggregates. Soil Sci Soc Am J
+              |       49(3):645-651 .
+              | 22.   Fischer TB, et al. (2008) Continuous time-resolved X-ray diffraction of the
+              |       biocatalyzed reduction of Mn oxide. American Mineralogist 93(11-12):1929-
+              |       1932 .
+              | 23.   Ahmed KM, et al. (2004) Arsenic enrichment in groundwater of the alluvial
+              |       aquifers in Bangladesh: an overview. Appl. Geochem. 19(2):181-200 .
+blank         | 
+              | 
+              | 
+meta          | 122
+ref           | 24.   McArthur J, Ravenscroft P, Safiulla S, & Thirlwall M (2001) Arsenic in
+              |       groundwater: Testing pollution mechanisms for sedimentary aquifers in
+              |       Bangladesh. Water Resour Res 37(1):109-117 .
+              | 25.   Fendorf S & Zasoski R (1992) Chromium(III) oxidation by delta-MnO2. 1.
+              |       Characterization Environ Sci Technol 26(1):79-85 .
+              | 26.   Masscheleyn P, Delaune R, & Patrick W (1991) Effect of redox potential and pH
+              |       on arsenic speciation and solubility in a contaminated soil Environ Sci Technol
+              |       25(8):1414-1419 .
+              | 27.   Jones C, Langner H, Anderson K, McDermott T, & Inskeep W (2000) Rates of
+              |       microbially mediated arsenate reduction and solubilization. Soil Sci Soc Am J
+              |       64(2):600-608 .
+              | 28.   Kocar BD, Herbel MJ, Tufano KJ, & Fendorf S (2006) Contrasting effects of
+              |       dissimilatory iron(III) and arsenic(V) reduction on arsenic retention and transport.
+              |       Environ Sci Technol 40(21):6715-6721 .
+              | 29.   Villaverde J, Van Beinum W, Beulke S, & Brown CD (2009) The Kinetics of
+              |       Sorption by Retarded Diffusion into Soil Aggregate Pores. Environ Sci Technol
+              |       43(21):8227-8232 .
+              | 30.   Sparks D (1989) Kinetics of soil chemical processes‎ (Academic Press, Inc., San
+              |       Diego, CA) p 210.
+              | 31.   Nesbitt H, Canning G, & Bancroft G (1998) XPS study of reductive dissolution of
+              |       7 angstrom-birnessite by H3AsO3, with constraints on reaction mechanism.
+              |       Geochim Cosmochim Ac 62(12):2097-2110 .
+              | 32.   Zhu M, Paul KW, Kubicki JD, & Sparks DL (2009) Quantum Chemical Study of
+              |       Arsenic (III, V) Adsorption on Mn-Oxides: Implications for Arsenic(III)
+              |       Oxidation. Environ Sci Technol 43(17):6655-6661 .
+              | 33.   Parikh SJ, Lafeerty BJ, Meade TG, & Sparks DL (2010) Evaluating
+              |       Environmental Influences on As-III Oxidation Kinetics by a Poorly Crystalline
+              |       Mn-Oxide. Environ Sci Technol 44(10):3772-3778 .
+              | 34.   Ginder-Vogel M, Landrot G, Fischel JS, & Sparks DL (2009) Quantification of
+              |       rapid environmental redox processes with quick-scanning x-ray absorption
+              |       spectroscopy (Q-XAS). P Natl Acad Sci USA 106(38):16124-16128 .
+              | 35.   Tournassat C, Charlet L, Bosbach D, & Manceau A (2002) Arsenic(III) oxidation
+              |       by birnessite and precipitation of manganese(II) arsenate. Environ Sci Technol
+              |       36(3):493-500 .
+              | 36.   Mayer K, Frind E, & Blowes D (2002) Multicomponent reactive transport
+              |       modeling in variably saturated porous media using a generalized formulation for
+              |       kinetically controlled reactions. Water Resour Res 38(9):1174 .
+              | 37.   Diem D & Stumm W (1984) ScienceDirect - Geochimica et Cosmochimica Acta :
+              |       Is dissolved Mn2+ being oxidized by O2 in absence of Mn-bacteria or surface
+              |       catalysts? Geochim. Cosmochim. Acta.
+              | 38.   Dzombak DA & Morel F (1990) Surface complexation modeling : hydrous ferric
+              |       oxide (Wiley, New York) pp xvii, 393 p.
+              | 39.   Mayer K, Benner S, Frind E, Thornton S, & Lerner D (2001) Reactive transport
+              |       modeling of processes controlling the distribution and natural attenuation of
+meta          |                                                                                       123
+ref           |       phenolic compounds in a deep sandstone aquifer. J Contam Hydrol 53(3-4):341-
+              |       368 .
+              | 40.   Laverman A, et al. (1995) Growth of strain SES-3 with arsenate and other diverse
+              |       electron-acceptors. Appl Environ Microb 61(10):3556-3561 .
+              | 41.   Luo Y & Millero F (2003) Solubility of rhodochrosite (MnCO3) in NaCl
+              |       solutions. J Solution Chem 32(5):405-416 .
+              | 42.   Garrels RM, Thompson ME, & Siever R (1960) Stability of Some Carbonates at
+              |       25-Degrees-C and One Atmosphere Total Pressure. Am. J. Sci. 258(6):402-418 .
+              | 43.   Morgan JJ ed (1967) Chemical equilibria and kinetic properties of manganese in
+              |       natural waters. (Wiley, New York), pp pp. 561-622.
+blank         | 
+              | 
+              | 
+              | 
+meta          | 124
+              | APPENDIX B: Supporting Information for
+              | Chapter 3
+blank         | 
+title         | B.1 Reactive Transport Modeling using MIN3P
+blank         | 
+text          |         The framework of our simulation is divided into a finite difference matrix
+              | consisting of three cells representing two chambers separated by a permeable membrane
+              | (representing the third cell). Dirichlet (fixed head) boundaries are set at both ends of the
+              | three-cell domain to simulate a closed system. The heads within both chambers remain
+              | identical at all times, therefore, there is no hydraulic gradient. Hence, hydraulic
+              | conductivities are not applicable in this system, and transport is solely governed by
+              | diffusion.
+              |         The biogeochemical conceptual model of our system is summarized in Table 1 of
+              | the main text, and can be divided into the following processes: 1) abiotic oxidation of
+              | As(III) by birnessite (eq. 1-3), 2) reductive dissolution of birnessite (eq. 4), 3)
+              | adsorption/desorption of As(V) and As(III) from goethite and birnessite (eq. 5-9).
+              | Arsenic adsorption/desorption is described using a simple reversible equilibrium
+              | expression formulated by Dzombeck and Morel (1) as described by Mayer et al. (2, 3).
+              | The diffusion coefficient was calibrated to As concentrations measured in chambers over
+              | time when As was injected in the absence of bacteria and birnessite.
+              |         Within the birnessite chamber cell, abiotic As(III) oxidation was described
+              | according to the general formulation:
+blank         | 
+text          |                                          " [Sm ] %Y
+              |                                           X
+              |                                 R = k[A] $(1)             '
+              |                                          # [K m ] + [Sm ] &(2)
+blank         | 
+text          |                Where Vmax is the maximum rate of reaction, term (1) represents the rate
+              | dependence on substrate concentration [A] (multiple substrates are allowed), with
+              | exponent X providing! reaction order (or fractional dependence), term (2) is a Monod
+              | expression with exponential dependence Y, where Sm represents a limiting substrate
+              | concentration and Km is a half-saturation constant.
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                         125
+title         |         Table B.1 Rate expressions for reaction used in simulation
+blank         | 
+              | 
+              | 
+text          | (Bio)geochemical      Rate Expression
+              | reaction
+              | As(III) Oxidation                                               #            [MnO 2 ]             & y1 #         [As(III)]            &y2
+              |                     R(As(III)-oxidation) = "k(As(III)-oxidation)% halfsat -[MnO 2 ]               ( % halfsat -[As(III)]              (
+              |                                                                 $ K(As(III)-oxidation) + [MnO 2 ] ' $ K(As(III)-oxidation) + [As(III)]'
+              |         * Kinetic and thermodynamic parameters given in table SI-2. All exponential values (yn)
+              |         are set to 1.
+              |         !
+blank         | 
+title         |         B.2 Diffusion coefficient calculations
+blank         | 
+text          |         Diffusion coefficient calculations used in this chapter are described in Appendix A.4.
+blank         | 
+              | 
+              | 
+              | 
+meta          |         126
+text          | Figure B.1 Diffusion of 480 !M As(III) (A) and As(V) (B) through semi-permeable
+              | polycarbonate membrane. Closed circles and opened circles indicate concentration of As in the
+              | input chamber and diffusion chambers, respectively.
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                          127
+text          | Figure B.2 Diffusion controlled sorption of As(III) or (V) on birnessite or goethite. One of the
+              | oxides was placed in a reaction chamber in As was then injected in the opposing chamber; the
+              | temporal changes in (top four panels) and sorbed (bottom four panel) As concentrations were then
+              | followed.
+blank         | 
+              | 
+meta          | 128
+title         | B.3 References cited in Appendix B
+ref           | 1.   Mayer K, Frind E, & Blowes D (2002) Multicomponent reactive transport
+              |      modeling in variably saturated porous media using a generalized formulation for
+              |      kinetically controlled reactions. Water Resour Res 38(9):1174 .
+              | 2.   Dzombak DA & Morel F (1990) Surface complexation modeling : hydrous ferric
+              |      oxide (Wiley, New York) pp xvii, 393 p.
+              | 3.   Mayer K, Benner S, Frind E, Thornton S, & Lerner D (2001) Reactive transport
+              |      modeling of processes controlling the distribution and natural attenuation of
+              |      phenolic compounds in a deep sandstone aquifer. J Contam Hydrol 53(3-4):341-
+              |      368 .
+blank         | 
+              | 
+              | 
+              | 
+meta          |                                                                                 129
+blank         |