Masters Theses

Date of Award


Degree Type


Degree Name

Master of Science


Environmental Engineering

Major Professor

Kevin Robinson

Committee Members

Kung-Hui Chu, Alice Layton


A thorough understanding of microbial dynamics in activated sludge treatment is currently unrealized due, in part, to quantitative techniques that rely on culture-dependent methods. The bias inherent with cultivation techniques may result in isolates that do not necessarily represent those dominant in the system evaluated. In situ analysis of microorganisms functioning in wastewater reactors will provide a greater understanding of how particular groups of organisms, such as nitrifiers, are impacted by system fluctuations. This information is vital for improved process design and control. The overall goal of this research was to investigate the use of the real-time polymerase chain reaction (PCR) technique as a tool for quantifying nitrifying bacteria in activated sludge. To facilitate evaluation of the real-time PCR assays over a range of nitrification performance efficiencies, operational parameters were manipulated to induce stress on the nitrifiers. The specific objective of this study was to assess the impact of operational and environmental changes on traditional measures of process performance and microbial cell densities of targeted microorganisms. Bench-scale activated sludge treatment units, consisting of four continuous-stirred tank reactors (CSTRs) operating in parallel, each with an external secondary clarifier, were operated to treat municipal wastewater collected from a full-scale wastewater treatment plant. The activated sludge reactors were operated at three constant temperature levels (20°C, 15°C, and 10°C) and three dissolved oxygen (DO) levels [high (≥ 2.0 mg/L), intermediate (1.0-1.5 mg/L) and low (≤0.5 mg/L) over a 21-month period. The reactors were operated at solids retention times (SRTs) of 20-, 10-, 5-, and 2-days. Treatment performance was determined by traditional physical/chemical parameters of chemical oxygen demand (COD) removal efficiency, mixed liquor volatile suspended solids (MLVSS) concentrations, and ammonia and nitrite oxidation rates. Real-time PCR assays were used to determine cell concentrations of total bacterial 16S rDNA, a gross measure of biomass content, the amoA gene of N. oligotropha-type bacteria, a measure of ammonia-oxidizing bacteria (AOB), and the Nitrospira 16S rDNA gene, a measure of nitrite-oxidizing bacteria (NOB). iii Efficient COD removal occurred throughout all phases of the study and carbon, however, carbon treatment performance was found to increase with increasing SRT. In contrast, DO concentration and temperature were not found to impact COD removal efficiency. SRT was found to be the prime determinant of both MLVSS and total eubacterial concentration and, as expected, the two parameters were positively correlated. Temperature and DO were not found to have a clear impact on either measure of total biomass. Consistent with the literature, the findings of this study indicated SRT was the single most important factor in determining whether or not nitrification will occur in a treatment system. Efficient nitrification occurred at low temperatures and low DO levels for the higher SRT reactors, but the 5-day and 2-day SRT reactors were dramatically affected by changes in these parameters. The contribution of N. oligotropha-type AOB to the total eubacteria population (

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