Graduation Year


Document Type




Degree Granting Department

Civil and Environmental Engineering

Major Professor

Peter G. Stroot, Ph.D.

Committee Member

James R. Mihelcic, Ph.D.

Committee Member

James R. Garey, Ph.D.

Committee Member

Scott W. Campbell, Ph.D.

Committee Member

Daniel H. Yeh, Ph.D.


Carbon Dioxide, AOB, NOB, Ammonium, FISH


Ammonium (NH4+) is a biological nutrient that is transformed in a wastewater treatment plant (WWTP) in a process called activated sludge. This is accomplished in an aerobic environment using microorganisms and inorganic carbon that convert the ammonium to nitrate (NO3-). This process is termed nitrification. Removal of ammonium is necessary due to its oxygen demand and toxicity to the environment.

Nitrification is considered a slow process due to the slow growth rate of the nitrifying bacteria. Ammonia oxidizing bacteria (AOB) first covert the ammonium (NH4+) to nitrite (NO2-) followed by conversion to nitrate (NO3-) by nitrite oxidizing bacteria (NOB). These slow rates limit the treatment capacity of the WWTP.

The initial hypothesis suggested that these slow rates were due to limited carbon in the aeration basin of a WWTP. A series of designed experiments and observational studies revealed substantial dissolved CO2 exists throughout a WWTP. Based on these findings, the central research focused on determining if an optimum dissolved CO2 concentration/ pH combination exists that maximizes nitrification.

Experimentation conducted at a pH of 7.0 and varying concentrations of dissolved CO2 concentration revealed inhibition at low (/l) and high (>30 mg/l) dissolved CO2 concentration levels. Further research found that optimum nitrification can be attained in a dissolved CO2 concentration range of 10 - 15 mg/l and a pH range of 7.5 - 8.0. A maximum specific growth rate of 1.05 - 1.15 days-1 was achieved. A partitioning of the sums of squares from these designed experiments found that pH accounts for approximately 83 percent of the sums of squares due to treatment with the dissolved CO2 concentration accounting for 17 percent. This suggests that pH is the dominant factor affecting nitrification when dissolved CO2 concentration is optimized.

Analysis of the growth kinetics for two of the designed experiments was conducted. However, a set of parameters could not be found that described growth conditions for all operating conditions. Evaluating the results from these two experiments may suggest that a microbial population shift occurred between 16 and 19 mg/l of dissolved CO2 concentration. These dissolved CO2 concentrations represent pH values of 7.1 and 7.0, respectively, and were compared to experimentation conducted at a pH of 7.0. Though the pH difference is minor, in combination with the elevated dissolved CO2 concentration, a microbial shift was hypothesized.

Microbial samples were collected from the designed experiment that optimized dissolved CO2 concentration (5, 10 and 15 mg/l) and pH (6.5, 7.0, 7.5 and 8.0). These samples were evaluated using Fluorescence in situ hybridizations (FISH) to determine the population density of common ammonium oxidizing bacteria (AOB) (Nitrosomonas and Nitrosospira) and nitrite oxidizing bacteria (NOB) Nitrobacter and Nitrospirae). The dominant AOB and NOB microbes were found to be Nitrosomonas and Nitrospirae.

These results suggest that increased nitrification rates can be achieved by incorporating appropriate controls in a wastewater treatment plant (WWTP). With higher nitrification rates, lower nitrogen values can be obtained which will reduce the WWTP effluent nitrogen concentration. Conversely, these increased nitrification rates can also reduce the volume of an aeration basin given similar effluent nitrogen concentrations.