Date of Award


Degree Type


Degree Name

Doctor of Philosophy


Civil Engineering

Major Professor

Kevin G. Robinson

Committee Members

Alice C. Layton, Gary S. Sayler, Gregory Reed, Paul Frymier


Reaction sequences of nitrifying and denitrifying bacteria are widely used to eliminate nitrogenous compounds from wastewater. During nitrification, ammonia is oxidized to nitrite by autotrophic ammonia oxidizing bacteria and nitrite is further oxidized to nitrate by nitrite oxidizers. Subsequently, nitrate or nitrite is reduced by denitrifying bacteria to gaseous nitrogen compounds. It is common knowledge that nitrification is an aerobic process and denitrification an anaerobic process. However, recent research has shown that denitrification can occur under aerobic conditions in pure cultures. Volatile fatty acids (VFAs), produced during anaerobic treatment processes, can affect both nitrite oxidation and aerobic denitrification. VFAs were shown to reduce nitrate formation via nitrite oxidation in activated sludge systems and to stimulate aerobic denitrification in pure cultures. Nitrite removal inhibition by VFAs observed in activated sludge systems may be due to the level of aerobic denitrification which occurs. Investigation of this possibility can provide a new insight for the removal of nitrogen from wastewater and possibly reduce the chemical and energy demand for nitrogen treatment. The overall goal of this research was to demonstrate that nitrification and denitrification could occur in the same reactor under aerobic conditions in the presence of VFAs.

The impact of VFAs on nitrite removal and nitrate formation in activated sludge systems was studied in batch and CSTR experiments. The experimental work included measurements of nitrite removal, nitrate formation and CO2 fixation in the absence and presence of VFAs. In addition, molecular tools were applied to examine changes in microbial population density when the population was exposed to VFAs. Production of N2O and activity of periplasmic nitrate reductase enzyme (NAP) which catalyses the first step of aerobic denitrification were also analyzed.

Nitrite removal and nitrate formation rates were reduced in the presence of VFAs in batch experiments. Nitrate formation rate was reduced to a greater extent (74%) than nitrite removal rate (35%) indicating that products other than nitrate were formed during nitrite oxidation. The addition of VFAs into an activated sludge CSTR treating municipal wastewater resulted in a rapid decrease in nitrate formation rate (> 70% reduction); however, nitrite removal rate was not reduced. No nitrogen was discharged in the effluent of the CSTR indicating that nitrogenous compounds were completely removed from the wastewater. In contrast, VFAs were not found to impact carbon dioxide fixation efficiency in either batch or CSTR experiments although it is generally believed to be limited by the availability of energy derived from nitrite oxidation. Non-inhibitory effect of VFAs on carbon dioxide fixation implied that VFAs disturb nitrite removal and nitrate formation by a different system other than nitrite oxidation. Additionally, the number of nitrite oxidizing bacteria (NOB) remained relatively constant in the presence of VFAs indicating that any reduction observed was not due to a decrease in NOB. N2O gas was produced in the presence of VFAs which was a clear indication that aerobic reduction of nitrite and/or nitrate occurred. It appeared that aerobic denitrification was responsible for the unbalanced nitrification conversions in the presence of VFAs. Also, the activity of NAP enzyme increased when VFAs were present suggesting a significant role of aerobic denitrification during nitrogen conversions.

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