Date of Award


Degree Name

Doctor of Philosophy


Molecular Biology, Microbiology and Biochemistry

First Advisor

Achenbach, Laurie


Humic substances (HS) are the humified portions of totally decomposed soil organic matter that are ubiquitous in nature. Although these substances have been studied for more than 200 years, neither their metabolic capabilities nor a specific chemical structure has yet to be determined. HS have been studied as a carbon source in many environments where they are degraded; however, previous studies have shown that some microorganisms are capable of utilizing humic substances as electron acceptors and electron donors in anaerobic respiration. Even though there have been humic-reducing and humic-oxidizing microorganisms isolated and studied in recent years, the mechanism of humics metabolism and its interaction in the natural environment are not well understood. However, it is known that the continuous change in the redox state of HS is important to the cycling of iron, stability of nitrogen and carbon, and the mobility and bioavailability of inorganic and organic environmental pollutants. In this study, microbial communities were examined to evaluate the community dynamics of nitrate-dependent HS-oxidizing populations and to provide a snapshot of the phylogenetic diversity of these microorganisms. Column studies were performed using nitrate as the sole electron acceptor and the following as the electron donors in different columns: reduced humic acids, oxidized humic acids, and acetate as the control. Liquid buffered media was added to a separate column to serve as an additional control. Polymerase chain reactions of the 16S rRNA genes using DNA from the column studies were performed and analyzed by constructing 16S rDNA clone libraries and by performing denaturing gradient gel electrophoresis (DGGE). Clones from the library have been sequenced and analyzed to paint a phylogenetic picture of the microbial community under the various conditions. Results indicate that the majority of the clones were assigned to four well-characterized divisions, the Acidobacteria, the Bacteroidetes, the Firmicutes, and the Proteobacteria. Additional findings suggest that members related to Bacteroidetes are involved in some sort of HS cycling in the environment or may be excellent electron scavengers enabling them to outcompete other microorganisms and that members of Proteobacteria may be the dominant HS-oxidizing microorganisms in natural environments. An additional aspect of this project hypothesizes that specific genes are differentially expressed when HS-oxidizing bacteria are growing on reduced HS as compared to acetate or in the presence of oxidized HS. To test this hypothesis, the global gene expression profile of Acidovorax ebreus strain TPSY was assessed using microarray analysis. This method led to the identification of several genes potentially involved in nitrate-dependent HS oxidization and a proposed model for this respiratory process in strain TPSY. The final section in this project was designed to assess in-service teachers' perceived levels of familiarity with and interest in learning more about selected microbiology concepts and their actual understanding of the selected microbiology concepts. Sixty-two in-service elementary, middle, and high school teachers from several school districts across southern Illinois completed a three-part instrument that included additional open-ended questions to gain more information about the teachers' conceptual understanding. The results of this study suggest that teachers who hold a teaching certification specific for teaching life science have taken more life science courses and scored significantly higher on the familiarity and conceptual knowledge sections of this study. The current research explores what is currently known about humic substances, specifically humics as an electron donor, analyzes the community structure in a humics oxidizing environment, identifies genes that are induced under nitrate-reducing, HS-oxidizing conditions, and evaluates the importance of microbiology to biological scientific literacy in today's society.




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