Date of Award

12-2002

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Gary S. Sayler

Committee Members

David R. Raman, Arthur J. Meyers, Steven W. Wilhelm, Jeffrey M. Becker

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.

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. 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.

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