MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Linduo Zhao Candidate for the Degree DOCTOR OF PHILOSOPHY ______________________________________ Hailiang Dong, Director ______________________________________ John Rakovan, Reader ______________________________________ Jonathan Levy, Reader ______________________________________ Christopher Gorski, Reader ______________________________________ Richard Edelmann, Graduate School Representative ABSTRACT IRON REDOX PROCESS IN CLAY MINERALS & ITS ENVIRONMENTAL SIGNIFICANCE by Linduo Zhao The importance of microbial nitrate-dependent Fe(II) oxidation to iron biogeochemistry is well recognized. Past research has focused on oxidation of aqueous Fe2+ and structural Fe(II) in oxides, carbonates, and phosphate, but the importance of structural Fe(II) in phyllosilicates in this reaction is only recently studied. However, the effect of clay mineralogy on the rate and the mechanism of the reaction, and subsequent mineralogical end products are still poorly known. The objective of the first research was to study the coupled process of microbial oxidation of Fe(II) in clay mineral nontronite (NAu-2), and nitrate reduction by Pseudogulbenkiania species strain 2002, and to determine mineralogical changes associated with this process. Bio-oxidation experiments were conducted using Fe(II) in microbially reduced nontronite as electron donor and nitrate as electron acceptor to investigate cell growth on this process. The bio-oxidation extent under growth and nongrowth conditions reached 67% and 57%, respectively. Over the same time period, nitrate was completely reduced. Abiotic oxidation by nitrite partly accelerated Fe(II) oxidation rate under the growth condition. The oxidized Fe(III) largely remained in the nontronite structure, but secondary minerals such as vivianite, ferrihydrite, and magnetite formed depending on specific experimental conditions. The objective of the second research was to study microbially mediated redox cycles of Fe in nontronite (NAu-2). During the reduction phase, structural Fe(III) in NAu-2 served as electron acceptor, lactate as electron donor, AQDS as electron shuttle, and dissimilatory Fe(III)-reducing bacterium Shewanella putrefaciens CN32 as mediator. During the oxidation phase, biogenic Fe(II) served as electron donor and nitrate as electron acceptor. Nitrate-dependent Fe(II)-oxidizing bacterium Pseudogulbenkiania sp. strain 2002 was added as mediator in the same media. For all three cycles, structural Fe in NAu-2 was able to reversibly undergo three redox cycles without significant dissolution. Fe(II) in bioreduced samples occurred in two distinct environments, at edges and in the interior of the NAu-2 structure. The overall objective of the third study was to study biological nitrate-dependent Fe(II) oxidation in illite IMt-1 and the effects of bio-oxidation on clay mineral transformation. Our data demonstrated that Pseudogulbenkiania sp. strain 2002 was able to couple oxidation of structural Fe(II) in IMt-1 with reduction of nitrate to N2 with nitrite as a transient intermediate. Fe(II)-oxidizing bacteria caused clay mineral structure change, and facilitated the illite→kaolinite and illite→smectite transformations. The biogenic smectite is a transient phase. IRON REDOX PROCESS IN CLAY MINERALS & ITS ENVIRONMENTAL SIGNIFICANCE A DISSERTATION Presented to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Geology and Environmental Earth Science by Linduo Zhao The Graduate School Miami University Oxford, Ohio 2015 Dissertation Director: Hailiang Dong © Linduo Zhao 2015 TABLE OF CONTENTS Chapter 1: Introduction ...................................................................................................1 References ...........................................................................................................................5 Chapter 2: Biological oxidation of Fe(II) in reduced nontronite coupled with nitrate reduction by Pseudogulbenkiania sp. Strain 2002 ...........................................................9 Abstract ..............................................................................................................................10 Body Text...........................................................................................................................11 References ..........................................................................................................................34 Tables and Figures .............................................................................................................42 Chapter 3: Biological redox cycling of iron in nontronite and its potential application in nitrate removal.........................................................................................56 Abstract ..............................................................................................................................57 Body Text...........................................................................................................................58 References ..........................................................................................................................73 Tables and Figures .............................................................................................................79 Chapter 4: Biological oxidation of Fe(II) in illite coupled with nitrate reduction and its role in clay mineral transform ..................................................................................99 Abstract ............................................................................................................................100 Body Text.........................................................................................................................101 References ........................................................................................................................114 Tables and Figures ...........................................................................................................117 Chapter 5: Conclusions and further recommendations .............................................129 iii LIST OF TABLES Chapter 2: Biological oxidation of Fe(II) in reduced nontronite coupled with nitrate reduction by Pseudogulbenkiania sp. Strain 2002 ..........................................................9 1- 77 K Fitting and calculated Mossbauer parameters......................................................42 Chapter 3: Biological redox cycling of iron in nontronite and its potential application in nitrate removal.........................................................................................56 1- Bio-reduction and bio-oxidation extents and initial rates ..............................................79 2- Fe L3 EELS peak position, the L3/L2 peak intensity ratios, and the comparisons of Fe(II) contents between the EELS and the chemical results ..............................................80 3- Calculated Mössbauer parameters for the room temperature spectrum of the 2nd bio- reduced NAu-2 sample with AQDS .................................................................................81 Chapter 4: Biological oxidation of Fe(II) in illite coupled with nitrate reduction and its role in clay mineral transformation ..........................................................................99 1- Fe(II) concentration before and after bio-oxidation, bio-oxidation extents and rates 117 iv LIST OF FIGURES Chapter 2: Biological oxidation of Fe(II) in reduced nontronite coupled with nitrate reduction by Pseudogulbenkiania sp. Strain 2002 ..........................................................9 1- Time-course changes of concentrations of Fe(II) in reduced NAu-2, nitrate, nitrite, protein (a proxy for biomass), and N2 produced from the nitrate-dependent Fe(II) oxidation using strain 2002 ................................................................................................46 2- X-ray diffraction patterns for bioreduced NAu-2 sample (i.e., onset of bio-oxidation) and for those oxidized by strain 2002 for various times (10, 30 and 60 days) under the growth (A) and the nongrowth conditions (B) ...................................................................47 3- Comparison of XRD patterns between abiotic control (e.g., bioreduced by strain CN32 cells but never oxidized by strain 2002 cells) and microbially oxidized samples after 60 days of inoculation of strain 2002 cells under the growth condition ................................48 4- X-ray diffraction patterns for the NAu-2 sample after reduction by CN32 cells for 2 weeks and oxidation by strain 2002 cells under the growth condition for 6 months. Vivianite and magnetite were detected in this sample ......................................................49 5- Secondary electron microscopic (SEM) images & energy dispersive spectroscopy (EDS) spectrum of NAu-2 samples after nitrate-dependent Fe(II)-oxidation of reduced NAu-2 by strain 2002 under the growth condition for 30 days .........................................50 6- SEM images of vivianite from the NAu-2 sample following reduction by CN32 cells for 2 weeks and oxidation by strain 2002 cells under the growth condition for 6 months ............................................................................................................................................51 7- Transmission