The common consensus among researchers and practitioners alike is that high free ammonia concentrations cause nitrite oxidizer inhibition in wastewater treatment systems. The work of Anthonisen et al. (1976), who produced an operational chart providing guidance on the free ammonia concentrations most likely to produce nitrite oxidizer inhibition, is considered by many to be the definitive work that established the inhibitory nature of free ammonia in wastewater treatment systems. Over the past three decades several investigators have reported discrepancies in the range of inhibitory free ammonia concentrations. Anthonisen et al. (1976) reported inhibition of nitrite-oxidizers at free ammonia concentrations of 0.1 to 1 mg/L, Turk and Mavinic (1986) reported no significant nitrite-oxidizer inhibition for free ammonia concentrations below 10 mg NH3-N/L, whereas Mauret et al. (1996) concluded that free ammonia inhibits Nitrobacter, in the range of 6.6 to 8.9 mg NH3-N/L. On the other hand, Gieseke et al. (2003) state that it is not known whether bacteria of the genus Nitrospira, now believed to be the most prevalent nitrite oxidizer in wastewater systems, are inhibited by free ammonia.
Some investigators have called into question the true cause of observed nitrite oxidizer inhibition. For example, Cecen and Ipek (1998) suggested that the dissolved oxygen to free ammonia ratio and not the free ammonia concentration itself is important, when attempting to induce nitrite accumulation. The work of others (Stuven et al. 1992; Yang and Alleman 1992; and Hyungseok Yoo et al. 1999) has suggested that hydroxylamine – an intermediate in the ammonia oxidation process – may be the true cause of nitrite oxidizer inhibition and therefore of nitrite accumulation. Many authors agree; however, that selective inhibition of nitrite oxidation via apparent free ammonia inhibition would allow direct oxidation of ammonia to nitrite and denitrification to dinitrogen gas with the inherent benefits of reduced aeration and carbon requirements. The starting point to designing such a process is the development of a clearer understanding of the mechanism(s) associated with nitrite oxidizer inhibition and nitrite accumulation.
This paper describes the results of exploratory work conducted to both confirm the results of mixed culture batch tests suggesting free ammonia may not be inhibitory to nitrite oxidizers as commonly reported and elucidate the likely mechanism(s) responsible for observed phenomenon. Free ammonia inhibition trials were undertaken using pure cultures of Nitrospira moscoviensis, since Nitrospira spp. were the predominant nitrite-oxidizers in the systems studied for this research program. The results of these experiments support the conclusion that free ammonia is probably not inhibitory to nitrite-oxidizing organisms in wastewater systems.
Several long-term reactor perturbation studies were conducted with mixed microbial cultures to identify both the population shifts and gas dynamics associated with apparent free ammonia inhibition phenomenon. These studies, like others conducted for this research program, showed that nitrous oxide emissions coincide with nitrite accumulation. Several experiments suggested autotrophic denitrification of nitrite by ammonia-oxidizing organisms as the most likely source. FISH analyses conducted for this research program showed that ammonia and nitrite-oxidizing organisms grew in colonies in close proximity to each other. Data is presented to suggest that the increase in oxygen utilization and denitrification of nitrite to nitrous oxide by ammoniaoxidizers following a free ammonia perturbation, results in reduced substrate availability for nitrite-oxidizers, and is the true cause of apparent free ammonia inhibition. A conceptual model explaining apparent free ammonia inhibition of nitrite-oxidizing organisms has been developed and is presented.
The work of Anthonisen et al. (1976) is considered by many to be the definitive work that established the inhibitory nature of free ammonia toward nitrite oxidizers. Anthonisen et al. (1976) hypothesized that, depending upon the operating conditions and initial loading rates, varying nitrite concentrations will persist without subsequent oxidation to nitrate, especially in solutions or wastewater having high organic nitrogen or ammonia-nitrogen concentrations. These authors postulated that inhibition was specifically related to free ammonia and free nitrous acid and that certain concentrations of free ammonia would inhibit nitrite but not ammonia oxidizers. In their original paper, they provide schematic representations of batch nitrification with and without inhibition. Anthonisen et al. (1976) used the associated concepts to develop an operational chart indicating the various concentration ranges where free ammonia and nitrous acid would likely be inhibitory to ammonia and/or nitrite oxidizers. These authors reported free ammonia was likely to be inhibitory towards nitrite oxidizers in the range 0.1 to 1 mg-NH3/L (0.08 to 0.8 mg NH3-N/L) and to ammonia oxidizers in the range 10 to 150 mg-NH3/L (8.2 to 123.5 mg NH3-N/L).
There has been a great deal of interest in free ammonia inhibition of nitrite-oxidizers since Anthonisen et al. (1976) published their seminal paper. Much of this interest has been driven by a desire to design nitrification/denitrification systems that selectively inhibit nitrite oxidation – thereby eliminating the formation of nitrate. Numerous authors (Voets et al. 1975; Turk and Mavinic 1986, 1987, 1989a, 1989b; Balmelle 1992; Chen et al. 1991; Fdz-Polanco et al. 1996; Garrido et al. 1997; Hyungseok Yoo et al. 1999) have reported the capital and operational benefits of the process, referred to here as the nitrate shunt, to include: a 25% reduction in aeration requirements, a 40% reduction in external carbon addition for denitrification in low C:N wastes, a potential reduction in anoxic zone volume and a significant reduction in sludge production.
Although free ammonia is still the consensus cause of nitrite oxidizer inhibition and therefore considered the key to process operation, there are discrepancies in reported inhibitory ranges, as well as the actual cause of observed nitrite oxidizer inhibition. For instance, Turk and Mavinic (1986, 1987, 1989a, 1989b) reported no significant nitrite-oxidizer inhibition for free ammonia concentrations below 10 mg NH3-N/L, whereas Mauret et al. (1996) concluded that free
ammonia inhibits Nitrobacter in the range of 6.6 to 8.9 mg NH3-N/L.
Several investigators have called into question the true cause of nitrite oxidizer inhibition. For example, Cecen and Ipek (1998) have suggested that the dissolved oxygen to free ammonia ratio, and not the free ammonia concentration itself, is of primary importance when attempting to induce nitrite accumulation. The work of others (Stuven et al. 1992; Yang et Alleman 1992; and Hyungseok Yoo et al. 1999) has suggested that hydroxylamine – an intermediate in the ammonia oxidation process – may be the true cause of nitrite oxidizer inhibition and therefore of nitrite accumulation.
In addition, it is now believed that Nitrospira, and not Nitrobacter, is typically the dominant nitrite oxidizer in most environmental matrices including wastewater systems (Schramm et al. 1998; Juretschko et al. 1998; Daims et al. 2000; Bartosch et al. 2002). This is in spite of the fact that described species of Nitrospira grow significantly slower in pure culture than Nitrobacter (Juretschko et al. 1998). Interestingly, Gieseke et al. (2003) state that it is not known whether bacteria of the genus Nitrospira are inhibited by free ammonia.
The work reported here was the direct result of the authors’ inability to reproduce nitrite oxidizer inhibition effects at free ammonia concentrations reported as inhibitory in the engineering literature. This work was carried out with a combination of mixed and pure cultures with the primary objectives of confirming whether or not free ammonia is truly inhibitory as well as the most likely cause(s) of the observed phenomenon. This paper summarizes the results of three specific experiments conducted as part of the overall research program and used to develop a conceptual model for apparent free ammonia inhibition. Pure cultures of Nitrospira moscoviensis were used in the first set of experiments to confirm free ammonia inhibition. This work was followed by mixed culture experiments with synthetic wastewater to study the response of mixed microbial populations to both a dissolved oxygen and free ammonia perturbation respectively.