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 D. Reed


A bench scale nitrification reactor (BSNR) containing nitrite oxidizing bacteria (NOB) of the genus Nitrobacter (» 1 × 1010/L; 5 - 22% of bacteria) was used to assess the ribosomal RNA transcript (rRNAt) to ribosomal RNA gene (rDNA) ratio as an in-situ nitrite oxidizing activity metric. A quantitative PCR detection system was developed that amplified a 143 base pair sequence within the Nitrobacter spp. 16S - 23S intergenenic spacer region. The resulting real-time PCR (rDNA) and real-time RT-PCR (rRNAt) assays were specific, accurate, and sensitive, requiring three replicate nucleic acid extracts to reliably detect a true 2-fold concentration difference in BSNR mixed liquor samples. These assays were used to establish upper (1.8) and lower (0.69) ratio prediction intervals (a = 0.10) for the BSNR operating at high nitrification efficiency. These intervals served as benchmarks when the ratio was measured in all subsequent experiments. It was hypothesized that during NOB inhibition the ratio would decline below 0.69 and during unlimited nitrite oxidation exceed 1.8.

When the Nitrobacter rRNAt/rDNA ratio was measured in a batch nitrite oxidation experiment, the metric value increased monotonically from a lower limit less than 0.10 during nitrite starvation to an upper limit greater than 3.0 eight hours after unlimited nitrite oxidation began. The ratio declined monotonically back to 0.10 within 4 hours of nitrite exhaustion. Because of the slow Nitrobacter grow rate, the rDNA concentration (» 6 ´ 109 copies/L) did not change appreciably. Thus, the metric response was mediated by rRNAt abundance that varied as the growth prospect changed from unfavorable (nitrite starvation: » 5 ´ 108 copies/L), to favorable (excess nitrite: » 3 ´ 1010 copies/L), and back to unfavorable (nitrite starvation: » 5 ´ 108 copies/L). This demonstrated that quantitative PCR is better suited to detect activity as opposed population changes in slow growing bacteria.

Batch inhibition experiments supported a hypothesized negative monotonic correlation between the Nitrobacter rRNAt/rDNA ratio and the inhibitor concentration and fraction inhibition level. For all the compounds studied, the ratio was significantly reduced (< 0.69) after 4.5 hours of exposure to concentrations that extensively inhibited nitrite oxidation. However, the response varied at the same inhibition level for the different inhibitors investigated. For 3,5-DCP (an uncoupler), the ratio dropped below 0.69 at concentrations that did not significantly reduce nitrite oxidation rates, suggesting that the metric is particularly sensitive to growth inhibitors. When this compound induced near complete inhibition of nitrite oxidation, the ratio dropped to » 0.06. For azide and H+, the ratio fell below 0.69 only if unlimited nitrite oxidation rates were reduced by ³ 80%. When nitrite oxidation was completely inhibited by a sudden pH change to 5.0, the ratio fell to » 0.3. A pH change to 4.5 also stopped nitrite oxidation, but the ratio did not decline. This indicated that the metric response can be disrupted if rRNAt processing is inhibited.

Though widely reported as an NOB inhibitor in the literature, free ammonia did not significantly reduce nitrite oxidation rates while ammonia oxidizing bacteria were inhibited. However, nitrite oxidation was significantly inhibited at low free ammonia concentrations if the ammonia oxidizing bacteria were allowed to remain active. This was accompanied by a low (» 0.10) rRNAt/rDNA ratio.

In a staged BSNR inhibition experiment, the Nitrobacter rRNAt/rDNA ratio accurately reflected nitrite oxidizing activity. The ratio dropped significantly (< 0.69) 22 hours before the nitrification efficiency fell below 90%. When nitrite oxidizing activity was later recovered, the ratio increased significantly (> 0.69) before this was apparent in the reactor soluble nitrogen data.

In summary, a wide range of experiments suggested the rRNAt/rDNA ratio was an effective monitor of in-situ nitrite oxidation activity. The metric could possibly be used to identify, remediate, and forecast nitrification and N-removal instability during wastewater treatment.

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