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Intrinsic Magnetism of Bacillus Spores: Theoretical Studies and Potential Applications

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Intrinsic Magnetism of Bacillus Spores: Theoretical Studies and Potential Applications Intrinsic Magnetism of Bacillus Spores: Theoretical Studies and Potential Applications DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jianxin Sun Ohio State Biochemistry Program The Ohio State University 2010 Dissertation Committee: Jeffrey J Chalmers, Advisor Jiyan Ma Shang-Tian Yang Copyright by Jianxin Sun 2010 Abstract Our lab was the first to report that Bacillus atrophaeus spores have intrinsic magnetism, and it was demonstrated that the magnetic susceptibility is sufficient to separate and deposit these spores on glass slides via a magnetic deposition system (Melnik et al. 2007). Surprisingly, Mn was the only paramagnetic element found in the spores, as indicated by Energy Dispersive Spectroscopy (EDS) analysis; but Mn is lacking in vegetative cells or other nonmagnetic spores. Moreover, magnetic susceptibility of different Mn oxides varies significantly, suggesting that the magnitude of a spore’s paramagnetism can differ based on a number of factors, including level of oxidation and time of sporulation. Experimentally, variation in the magnitude of paramagnetism was observed from batch to batch, and studies were conducted to further understand this variation including elemental quantification, optimization of the culturing, sporulating condition, and genetic studies. By adding polystyrene microparticles, which are of a very different size and magnetic susceptibility than spore clusters, the accuracy of Cell Track Velocitimetry (CTV) to measure and calculate magnetic susceptibility was explored and is reported. With the combination of CTV and X-ray Photoelectron Spectra, the oxidation state of Mn on spores and its imparting magnetic susceptibility to the spores was quantified, and the ii Mn per spore cluster was calculated. The calculation was consistent with the result from Inductively Coupled Plasma- Mass Spectrometry independent measurements. Different metal ion concentrations, oxygen availability and heat shock effects were examined during fermentation and Mn concentration was found to be the only factor that influenced the paramagnetism. Using these optimization studies, the Mn concentration was doubled in the media and the fermentation was scaled-up to 4 L using Biostat B bioreactor, and reliable magnetic spores were obtained. Potential applications of paramagnetic spores were also discussed: separation from various liquid food and adsorption of heavy metal ion from waste water. The intrinsic magnetism enabled the spores to be separated from air or liquid food, such as milk and chicken broth. However, spores couldn’t be separated from juice probably due to the low pH, in which the spores lost their paramagnetism. Interesting, the magnetism of spores recovered after adding Mn. Since spores could survive a sterilization process, the effect of sterilization on spores’ magnetism was examined. It turned out that the process didn’t alter the magnetic susceptibility of spores dramatically and surviving spores could be separated through the magnetic deposition system. iii Dedication Dedicated to my parents iv Acknowledgment I would like to thank Dr. Jeffrey Chalmers, my adviser, for his guidance, support and enthusiasm during the whole development of my research, which made the dissertation possible. I would like to thank Dr. Zborowski, who helped me with my papers and also theoretical questions during my study. I would like to thank my committee members for their suggestions on my dissertation. I would like to express my appreciation for Lisa in Evans Lab helping me with X-ray Photoelectron Spectrometry. I am grateful to Dr. Yang Zhao for his help in CTV adjustment and improvement all the time, to Priya and Brian for teaching me how to use fluorescent microscope and associated software. In addition, I would like to thank my friends, especially Dr. Yali Zhang and Ching-suei Hsu, for their suggestions, Dr. Wei-lun Chang, for her help in writing, Dr. Mingrui Yu and Derek Lyttle for proofreading. Finally, I would say thank you to my parents. I couldn’t make it without their encouragement. v Vita March, 1982....................................................Born, Hebei, China June, 2004 ......................................................B.S. Biological Science, Nankai University, China September, 2004-December, 2009...................M.S. Applied Statistics The Ohio State University September, 2004-September, 2010 ..................Graduate Research Associate, Ohio State Biochemistry Program The Ohio State University Publications Melnik, K., Sun, J., Fleischman, A., Roy, S., Zborowski, M. and Chalmers, J.J. (2007). Quantification of magnetic susceptibility in several strains of Bacillus spores: Implications for separation and detection. Biotechnology and Bioengineering 98(1): 186- 192. Fields of Study Major Field: Ohio State Biochemistry Program Specialization: Biochemical Engineering vi Table of Contents Abstract............................................................................................................................... ii Dedication.......................................................................................................................... iv Acknowledgment ................................................................................................................ v List of Tables ................................................................................................................... xiv List of Figures................................................................................................................. xvii Chapter 1 Introduction ........................................................................................................ 1 1.1 Biological Warfare (BW) and detection challenges.................................................. 1 1.2 Detection and separation of Bacillus atrophaeus spores .......................................... 4 1.3 Other Bacillus strains in the study ............................................................................ 7 1.4 Bacillus spores, Mn and paramagnetism................................................................... 8 1.5 Immunomagnetic Separation (IMS)........................................................................ 12 1.5.1 Introduction of IMS.......................................................................................... 12 1.5.2 Commercial Immunomagnetic Separation Technologies................................. 14 1.5.3 Magnetic deposition system ............................................................................. 15 1.6 Intrinsic magnetism................................................................................................. 16 vii 1.7 Magnetic susceptibility and magnetophoretic mobility .......................................... 19 1.7.1 Magnetic susceptibility..................................................................................... 19 1.7.2 Magnetophoretic Mobility (MM) ..................................................................... 21 1.8 Organization of the dissertation .............................................................................. 24 Chapter 2 Quantification of magnetic susceptibility in several strains of Bacillus spores27 2.1 Introduction ............................................................................................................. 28 2.2 Materials and methods ............................................................................................ 31 2.3 Results ..................................................................................................................... 37 2.4 Discussion ............................................................................................................... 45 Chapter 3 Improvement of Cell Tracking Velocimetry (CTV) accuracy by the inclusion of internal control in spore samples.................................................................................. 48 3.1. Motivation .............................................................................................................. 48 3.2. Theory .................................................................................................................... 51 3.2.1 Fundamental forces, velocities of particles in CTV ......................................... 51 3.2.2 Algorithm of CTV and classification by CTV associated software ................. 54 3.2.3 Classification by Support Vector Machines (SVMs) ....................................... 54 3.3 Materials and methods ............................................................................................ 54 3.3.1 Experimental apparatus .................................................................................... 54 3.3.2 Spores and particles.......................................................................................... 56 viii 3.4 Result....................................................................................................................... 60 3.4.1 Classification by 2 methods.............................................................................. 60 3.4.2 Magnetic susceptibility measurements of Polystyrene Microspheres (PSM) .. 61 3.4.3 Magnetic susceptibility of measurements and calculations of the spores ........ 65 3.5 Conclusion............................................................................................................... 73 Chapter 4 Quantification of both the presence, and oxidation
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