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Respiratory Pathways Used by Perchlorate–Respiring

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Respiratory Pathways Used by Perchlorate–Respiring The Pennsylvania State University The Graduate School College of Engineering RESPIRATORY PATHWAYS USED BY PERCHLORATE–RESPIRING BACTERIA A Thesis in Environmental Engineering by Yanguang Song ©2004 Yanguang Song Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2004 The thesis of Yanguang Song was reviewed and approved* by the following: Bruce E. Logan Kappe Professor of Environmental Engineering Thesis Advisor Chair of Committee William D. Burgos Associate Professor of Environmental Engineering John M. Regan Assistant Professor of Environmental Engineering Mary Ann Bruns Assistant Professor of Soil Science/Microbial Ecology Andrew Scanlon Professor of Civil Engineerin g Head of the Department of Civil and Environmental Engineering *Signatures are on file in the Graduate School. ABSTRACT Systematic experiments were performed to examine the inhibitory effects of oxygen on perchlorate reduction and to identify the components and branches in the electron transport chains involved in perchlorate reduction and aerobic respiration by Dechlorosoma sp. KJ (ATCC strain BAA-592). To investigate the effect of dissolved oxygen (DO) on perchlorate reduction, anaerobically grown cultures of Dechlorosoma sp. KJ were exposed to near-saturated concentration of DO for various periods of time. It was determined that cells exposed to oxygen for more than 12 hours were incapable of reducing perchlorate. Cells exposed to oxygen for less than 12 hours, when re-introduced to anoxic conditions, quickly reduced the redox potential to highly negative values (-127 mV to -337 mV) and were able to reduce perchlorate or chlorite. This result suggested that aeration during backwashing of biofilm reactors, or exposure of perchlorate-degrading cell suspensions to oxygen for periods of less than 12 hours, will not be detrimental to the ability of perchlorate-degrading bacteria to use perchlorate as an electron acceptor. Inhibitors were used to block specific respiratory enzymes, and thus to identify elements of the electron transport chain (ETC) involved in the reduction of molecular oxygen and perchlorate (Chapters 3 and 4). Inhibition of dissimilative perchlorate reduction by a low concentration of cyanide was due to oxygen build up, not inhibition of enzymes used for perchlorate reduction. The threshold DO concentration to inhibit perchlorate reduction was < 0.04 mg/L. The main ETC used by cells grown on high DO concentrations likely contains cytochrome bc1, cytochrome c and cytochrome aa3. When cells were grown on perchlorate, DO uptake occurred via a shorter ETC than that used by cells grown at higher DO concentrations. The ETC to oxygen at low DO concentrations is branched off the quinone to cytochrome bd, while the ETC to perchlorate is branched off the quinone to perchlorate reductase. These results should be useful in helping to improve the design and operation of biological perchlorate treatment systems. iii TABLE OF CONTENTS LIST OF TABLES ...............................................................................................................vii LIST OF FIGURES.............................................................................................................viii ACKNOWLEDGEMENTS.....................................................................................................x 1. INTRODUCTION..............................................................................................................1 1.1 Problem Statement and Research Objectives ............................................................1 1.2 Literature Review...................................................................................................2 1.2.1 Perchlorate respiring bacteria (PRB)................................................................2 1.2.2 Perchlorate degradation pathway .....................................................................5 1.2.3 ETC in PRB...................................................................................................7 1.3 Organization of this Dissertation..............................................................................8 1.4 References ...........................................................................................................11 2. Effect of O2 Exposure on Perchlorate Reduction by Dechlorosoma sp. KJ............................18 Abstract...........................................................................................................................18 2.1 Introduction .........................................................................................................19 2.2 Material and methods ............................................................................................21 2.2.1 Bacterium and media ....................................................................................21 2.2.2 Preparation of cell suspensions ......................................................................22 2.2.3 Oxygen scavengers .......................................................................................22 2.2.4 Dissolved oxygen probe measurements of oxygen removal rates......................22 2.2.5 Effect of different oxygen scavengers on cell growth ......................................23 2.2.6 O2 exposure procedures.................................................................................23 2.2.7 Analytical techniques....................................................................................24 2.3 Results.................................................................................................................24 2.3.1 Oxygen scavengers .......................................................................................24 2.3.2 Redox potential ............................................................................................25 iv 2.3.3 Effect of oxygen exposure on redox potential change ......................................25 2.3.4 Effect of oxygen exposure on perchlorate reduction ........................................26 2.4 Discussion............................................................................................................27 2.5 Conclusions .........................................................................................................29 2.6 Acknowledgement ................................................................................................30 2.7 References ...........................................................................................................31 3. Cyanide Inhibition of Enzyme Activities Involved in Perchlorate Reduction .........................40 Abstract...........................................................................................................................40 3.1 Introduction .........................................................................................................41 3.2 Material and methods ............................................................................................42 3.2.1 Bacterium and media ....................................................................................42 - 3.2.2 Culture growth in the presence of CN ............................................................43 3.2.3 Accumulation of DO during anaerobic cell respiration ....................................44 - 3.2.4 Perchlorate culture growth with CN and chemical oxygen scavengers.............44 - 3.2.5 Chlorite dismutation in the presence of CN ....................................................45 3.2.6 Analytical Techniques...................................................................................45 3.3 Results.................................................................................................................46 - - 3.3.1 CN inhibition of cell growth on ClO4 ............................................................46 - - 3.3.2 Culture growth on ClO3 or O2 in the presence of 1 mg/L of CN ......................47 - 3.3.3 DO accumulation in anaerobic cultures treated with CN .................................47 - 3.3.4 Perchlorate culture growth in the presence of CN and oxygen scavengers........48 - 3.3.5 Chlorite dismutation with different concentrations of CN ...............................49 3.4 Discussion............................................................................................................49 3.5 Acknowledgement ................................................................................................50 3.6 References ...........................................................................................................51 4. Different Electron Transport Chains to Molecular O2 in Perchlorate-Respiring Bacteria ........58 v Abstract...........................................................................................................................58 4.1 Introduction .........................................................................................................60 4.2 Material and methods ............................................................................................61 4.2.1 Bacterium and media ....................................................................................61 - 4.2.2 Effect of different levels of dissolved oxygen on ClO4 ....................................62 4.2.3 Aerobic and perchlorate reduction in the presence of respiratory inhibitors.......63 4.2.4 Cytochromes assays......................................................................................64
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