Date of Award


Degree Type


Degree Name

Doctor of Philosophy


Civil Engineering

Major Professor

Kevin G. Robinson

Committee Members

Chris D. Cox, Gary S. Sayler, Alice C. Layton, Arthur J. Meyers


The population size and physiological activity of ammonia-oxidizing bacteria (AOB) are two crucial rate-limiting parameters for the removal of ammonia from wastewater (Wagner et al., 1995). These two biological parameters may be affected by operational/environmental conditions (e.g., low pH, low temperature, low dissolved oxygen (DO), or short solids retention time (SRT)), and these impacts may subsequently influence ammonia removal performance in wastewater treatment systems (Okabe et al., 1996; Nogueira et al., 2002; Robinson et al., 2004; Zimmerman et al., 2004; Hallin et al., 2005). The impacts of changes in population dynamics and microbial activity of AOB on overall nitrification efficiency during wastewater treatment have not been fully addressed. The fundamental goal of this study was to assess AOB population dynamics (based on gene amoA measurement) and AOB physiological activity (based on amoA mRNA transcript measurement) as a function of changes in operational conditions (i.e., SRT, NH3, and, DO), and relate these molecular parameters to overall ammonia oxidation treatment performance.

This study consisted of three components to achieve the overall research goal. Component I focused on estimating long-term shifts in AOB population dynamics under different operational conditions (four SRTs, two NH3 levels, and two DO levels) in an industrial wastewater (bench-scale activated sludge) treatment system. Results suggested that the AOB population dynamics in this treatment system were primarily influenced by SRT and NH3 levels. Short-term changes in NH3 concentration affected AOB activity (based on ammonia oxidation rates); however, with long-term changes in NH3 level, the AOB population gradually shifted in short SRT systems due to ammonia induced selection of different AOB strains. Changes in DO concentration (from 3 mg/L to 0.5 mg/L) did not significantly affect AOB population dynamics. In general, overall AOB population levels did not rapidly respond to the tested operational changes. Thus, real-time fluctuations in the overall ammonia oxidation performance of the treatment system were not solely reflected by AOB population levels.

In research components II and III, amoA mRNA abundance was measured to determine whether this metric was related to AOB activity. In component II, an AOB culture from a laboratory enriched nitrifying system was used to examine the amoA mRNA response under varying NH3 and DO levels to assess AOB physiological activity at the transcriptional level. Results indicated that amoA mRNA abundance quickly (few hours) responded to different ammonia levels while amoA DNA levels did not notably change; and, the maximum amoA mRNA level reflected the ammonia oxidation activity of AOB cells under each ammonia condition. However, the amoA mRNA level did not correspond to the significant drop in ammonia oxidation activity during DO limitation. In component III, the physiological activity of each AOB population in the industrial bench-scale system was assessed based on amoA mRNA measurements to determine if activity ascendancy of an AOB strain lead to its population dominance in the treatment system under two different ammonia loadings. Results indicated that the dominant AOB population had relatively higher amoA mRNA abundance than the other strain’s allowing this group to be more active than other AOB groups.

In summary, the physiological activity measurements, based on amoA mRNA assessment, were responsive to some changes in operational conditions (i.e., varying NH3 concentrations at sufficient oxygen levels). Thus, this would allow quick identification of fluxes in ammonia oxidation performance for nitrifying systems. However, shifts in ammonia oxidation rate did not strictly correlate to changes in amoA mRNA level suggesting that AOB oxidation activity involved additional regulatory mechanisms other than amo expression, possibly at the post-transcriptional level. The amoA mRNA assessment may reflect an AOB oxidation activity shift in some cases but cannot be solely used to evaluate ammonia oxidation performance in all nitrifying systems. Hence, traditional oxidation-rate-based estimation is still necessary for routinely monitoring ammonia oxidation performance in treatment systems. Nevertheless, the biological parameters of AOB population levels and cellular activity provide insight into the microbial processes behind nitrification thereby enhancing our understanding of the nitrogen conversion process.

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