Antibody assessment of nitrifiers
Abstract
This dissertation describes the development of cell surface-targeted antibody probes for the identification of Nitrosomonas. The target microorganism is an autotrophic ammonia-oxidizer and belongs to one of nine known genera of nitrifying bacteria. It plays a central role in the global nitrogen cycle, and as such, is an important biological resident throughout the ecosystem, including: soils, streams, lakes, rivers, estuaries and oceans, as well as man-made wastewater treatment plants. As the only genus of ammonia-oxidizing bacteria known to exist in sewage sludge, its role in wastewater nitrification is unquestionable. Traditional culture techniques for determining the presence of these bacteria are severely limited by their slow growth rates, to the point where a standardized means for their enumeration has never been established. Experimental work on the development of an antibody-based procedure for their measurement was initiated a few decades ago by various researchers using fluorescence as a quantitative parameter. However, this method was found to be unduly complicated with background interference. This research, consequently, focused on the identification of Nitrosomonas with enzyme-labeled antibodies. The development of polyclonal antibodies against the type strain, Nitrosomonas europaea (ATCC 19718), successfully demonstrated the fundamental utility of an enzyme-linked immunosorbent assay (ELISA) protocol. Following an initial purification step to enhance overall specificity, this test had an apparent lower limit of detection of ${\sim}10\sp6$ cells per mL. Tests conducted with activated sludge samples exhibited a distinct difference between nitrifying and non-nitrifying mixed liquors, although the highest Nitrosomonas levels observed (i.e. at 1 to 2% of the overall cell density) were relatively close to the latter detection boundary. Monoclonal antibody development for two sewage strains of Nintrosomonas (Woods Hole strains C-91 and C-101) resulted in the isolation of antibody producing cell lines with varying degrees of specificity for soil strains as well. Monoclonal antibodies for freshwater strains of Nitrosomonas could not be isolated in this research. Altogether 30 cell lines, selected on the basis of their specificity for Nitrosomonas, were isolated and frozen. Their application to real world testing will require further characterization of the monoclonal antibodies.
Degree
Ph.D.
Advisors
Alleman, Purdue University.
Subject Area
Civil engineering|Environmental science|Microbiology|Biogeochemistry|Sanitation
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