The primary objective of this study was to track reactor gas emissions and microbial population dynamics during the acclimation of a bench scale SBR system operating via the nitrate shunt, defined here as the oxidation of ammonia to nitrite with denitrification of nitrite to nitrogenous
gases, to elucidate potential strategies for overcoming the acclimation response. Aerobic phase nitrite accumulation was induced by periodically subjecting the nitrite oxidizer population to an expected inhibitory concentration of free ammonia under anoxic conditions as previously described
by Turk and Mavinic (1986, 1987, 1989a, 1989b). A lab scale sequencing batch reactor (SBR) was operated with a pre-anoxic fill and high pH to subject nitrifiers to free ammonia concentrations reported to be inhibitory during the start of the aerobic phase. The anoxic period pH (and therefore
free ammonia concentration) was controlled by adjusting influent feed pH via a combination of sodium hydroxide and sodium bicarbonate addition. This was the same pH control scheme used by others (Turk and Mavinic (1986, 1987, 1989a, 1989b)) who have successfully operated systems via the nitrate
shunt. The target anoxic zone free ammonia concentration was 8-to-10 mg NH3-N/L. The aeration rate was initially kept constant and the aerobic phase dissolved oxygen level was used as a gauge of nitrifier health. Tracking studies were conducted during several SBR cycles both
during the shunt period and following the collapse of the shunt (acclimation), in order to elucidate the differences in process response, over and above nitrite accumulation. Changes in reactor microbial populations were tracked using a combination of slot blot hybridization and fatty acid
analyses. The control of anoxic free ammonia concentration through influent pH adjustment resulted in nitrite accumulation followed by eventual process acclimation and complete nitrite oxidation. Nitrite oxidation was initiated at free ammonia concentrations far in excess of the inhibitory
range reported by Anthonisen et al. (1976). By the time the shunt had collapsed, nitrite oxidation was initiated at free ammonia concentrations exceeding 9 mg-N/L. The period of nitrite accumulation was coincidental with reactor nitrous oxide emissions. RNA and suspended solids data
suggest that the entire nitrifier population was severely compromised when the initial feed pH adjustment was made to increase free ammonia concentration and initiate the nitrate shunt. The drop in RNA binding nitrifier molecular probes (Nso 190, Ntspa 454, Ntspa 685 and Nb 1000 probes) coincided
with an increase in effluent suspended solids. A drop in RNA concentration of this magnitude suggests the death and lysis of cells. The drop in total RNA suggested the entire population was compromised; however, the nitrite oxidizer population as measured by the Ntspa 454, Ntspa 685 and Nb
1000 RNA probe concentrations was most affected. The establishment of a new suspended solids equilibrium in the reactor supports the possibility that the change in pH may have changed overall process yield, due to a reduction in the ability to produce a proton motive force and/or a nutrient
limitation brought on by the high pH. The initial pH perturbation rather than free ammonia inhibition was shown to be the initial cause of nitrite accumulation and the shunt period was simply the result of the lower yield of nitrite oxidizers relative to ammonia oxidizers during the overall
recovery period. The observed acclimation of the nitrite oxidizer population was the result of the proliferation of small populations, following the original pH perturbation and the decrease in nitrite oxidizer substrate availability via the denitrification of nitrite to nitrous oxide by ammonia-oxidizers.
Reactor nitrous oxide emissions declined as the nitrite-oxidizer population recovered and was able to out compete ammonia-oxidizers for nitrite (ammonia oxidizers are able to denitrify nitrite to nitrous oxide). The nitrous oxide emissions were essentially zero following complete nitrite oxidizer
recovery. The research illustrates the value of molecular methods when interpreting microbial phenomenon in wastewater systems. It also points to the potential value of using reactor nitrous oxide emissions monitoring for gauging nitrifier and specifically nitrite-oxidizer function and
health. This research is also part of a growing body of evidence suggesting that free ammonia is not inhibitory to nitrite-oxidizers in wastewater systems as reported in the literature for the past thirty years.
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