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Isolation and Investigation of the Exopolysaccharide from Thauera Sp University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2002 Isolation and Investigation of the Exopolysaccharide from Thauera sp. MZ1T Michael S. Allen Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Medical Microbiology Commons Recommended Citation Allen, Michael S., "Isolation and Investigation of the Exopolysaccharide from Thauera sp. MZ1T. " PhD diss., University of Tennessee, 2002. https://trace.tennessee.edu/utk_graddiss/2089 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Michael S. Allen entitled "Isolation and Investigation of the Exopolysaccharide from Thauera sp. MZ1T." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Microbiology. Gary S. Sayler, Major Professor We have read this dissertation and recommend its acceptance: David R. Raman, Arthur J. Meyers, Steven W. Wilhelm, Jeffrey M. Becker Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a dissertation written by Michael S. Allen entitled “Isolation and Investigation of the Exopolysaccharide from Thauera sp. MZ1T.” I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Microbiology. Gary S. Sayler Major Professor We have read this dissertation And recommend its acceptance: David R. Raman Arthur J. Meyers Steven W. Wilhelm Jeffrey M. Becker Accepted for the Council: Anne Mayhew Vice Provost and Dean of Graduate Studies (Original signatures are on file with official student records.) Isolation and Characterization of the Exopolysaccharide Produced by Thauera strain MZ1T and Its Role in Flocculation A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Michael S. Allen December 2002 ACKNOWLEDGEMENTS I would first like to thank my major professor and mentor, Gary Sayler, for his guidance and support throughout this research. I would also like to thank the other members of my committee, Art Meyers, Steve Wilhelm, Raj Raman, Gary Stacey, and Jeff Becker, for their patience and support. I would particularly like to thank Art Meyers for his support and assistance above and beyond the normal call of duty. I am very grateful to David Baker for his assistance and insight, and to his students Karen Welch and Benjamin Prebyl. I am also grateful to Curtis Lajoie, Alice Layton, and Anthony Hay for their assistance early on in this project. I would also like to thank David Nivens for his time, thoughts, and assistance on a variety of aspects of this research, and to all the members of the CEB, each of who have assisted me at some point along the way. I would also like to thank my friends James Rice, Scott Moser, Chris Elkins, Cathy Scott, Dan Williams, and Victoria Garrett for their continued patience and unwavering moral support. Finally, I would like to thank Amy Tomaszewski to whom I owe a great deal beyond what is contained in this manuscript. ii ABSTRACT Thauera sp. strain MZ1T is a floc-forming bacterium isolated from the wastewater treatment plant of Eastman Chemical Company. Its overabundance in that system in the form of zoogloeal clusters was positively correlated to episodes of poor dewatering of activated sludge (Lajoie 2000). The specific cause of this problem was thought to be due to the production of large quantities of hydrophilic exopolysaccharide (EPS) by MZ1T, which entraps water in the form of a hydrated gel, and results in a sludge that is resistant to mechanical dewatering. A method for the reproducible extraction of EPS from pure cultures of MZ1T was developed. Subsequent investigation of the physical properties of the purified EPS found that the polymer was highly soluble in water but insoluble in non-aqueous solvents. The polymer was also found to be thermally stable. Investigations into the interaction of the EPS with metal cations revealed that the EPS showed a capacity for binding uranium ions in both aqueous and non-aqueous solutions. Additionally, the EPS was found to interact with calcium chloride, resulting in the precipitation of the EPS from solution. The glycosyl composition of the EPS was determined by gas chromatography– mass spectroscopy (GC–MS) of both the alditol acetate and per-O-trimethylsilyl methyl glycoside (TMS) derivatives. By these methods, the EPS was found to include: rhamnose, N-acetylfucosamine, galacturonic acid, N-acetylglucosamine, and trace amounts of glucose. iii Spectroscopic analyses of MZ1T EPS by one- and two-dimensional nuclear magnetic resonance (NMR) and Fourier-transform infrared (FT–IR) spectroscopy were used to support the chemical analyses and to identify the probable linkage of monosaccharides within the polymer and their respective D- or L- configurations. Spectroscopic analyses also revealed the presence of an aglycon substituent present on the EPS polymer. While all the monosaccharides detected in the chemical analyses could similarly be identified in NMR spectra, no through-space interactions were detected between glucose and any of the other monosaccharides. These results, along with data indicating the presence of glucose in gel permeation column fractions in the absence of other monosaccharides, suggest that glucose may be present in the form of a second polysaccharide in the EPS preparations. Several mutants of MZ1T incapable of, or reduced in, their capacity for floc formation in liquid media were isolated following chemical mutagenesis. Investigations of EPS extracted from these mutants revealed that all of the monosaccharides previously detected in the wild type EPS could also be identified in the EPS of the mutants, indicating that loss of floc forming capacity was not a result of alteration in the glycosyl composition of the EPS. Spectroscopic analysis by FT–IR of the EPS extracted from true floc– mutants did, however, reveal conserved alterations in the spectra of the mutant EPS relative to that from the wild type. These data suggest that alteration of the linkage or the substitution of the EPS is responsible for the loss of floc-forming capacity in the mutants. Additionally, it was found that floc– and floc-reduced isolates, unlike the wild type, were competent to receive broad host range plasmids by conjugal transfer. iv Colonies of these mutants also exhibited altered colony texture and differential responses to stains and dyes than did the wild type. Taken together, these data suggest a protective role of the EPS of MZ1T, and that mutations resulting in alterations of the EPS broadly affect the cell surface organization, intercellular interactions, and floc-forming capacity. v TABLE OF CONTENTS I. Introduction……………………………………………………………1 II. Literature Review……………………………………………………...5 a. Activated sludge wastewater treatment systems…………………..5 b. Flocculation-associated problems in the activated sludge system...6 c. Sludge dewatering…………………………………………………8 d. Theory of biological floc formation……………………………….8 e. Mechanisms of biological flocculation ………………………….10 f. EPS biosynthesis…………………………………………………12 g. Roles of EPS in nature…………………………………………...15 h. Factors influencing EPS production……………………………..16 i. Zoogloea…………………………………………………………17 j. The genus Thauera………………………………………………21 k. Goals of this research…………………………………………….27 III. Materials and Methods…………………………………….…………32 a. Bacterial plasmids and strains……………………………………32 b. Culture conditions and storage…………………………………...32 c. Media and chemicals……………………………………………..34 d. Molecular Biology Techniques…………………………………..36 e. DNA sequencing…………………………………………………37 f. BOX polymerase chain reaction (PCR) …………………………37 g. Ribosomal intergenic sequence analysis (RISA).………………..37 h. Determination of doubling time………………………………….38 i. Carbon-source utilization………………………………………...38 j. Carbohydrate concentration determination………………………39 k. Protein concentration determination……………………………..41 l. Isolation and purification of exopolysaccharide…………………41 m. Size Determination of EPS polymer……………………………..43 n. Physical properties of MZ1T EPS……………………………….43 o. Metal binding by EPS……………………………………………44 p. Alditol acetates…………………………………………………...45 q. Per-O-trimethylsilyl methyl glycosides (TMS) …………………47 r. Gas chromatography–mass spectrometry (GC–MS) ……………51 s. Nuclear Magnetic Resonance (NMR) Spectroscopy…………….51 vi t. Fourier-transform infrared spectroscopy (FTIR).………………..52 u. Conjugation ……………………………………………………...52 v. Electroporation.…………………………………………………..53 w. Mutagenesis.……………………………………………………..54 x. Floc+/– screening assays………………………………………….54 y. MZ1T-39A transposon mutagenesis and mapping………………57 IV. Results………………………………………………………………..60 a. Isolation and identification of MZ1T…………………………….60 b. Microscopic examination of MZ1T.…………………………..…60 c. Growth rates and doubling times of MZ1T and selected floc–
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