Aerobic ammonium oxidizing bacteria were first isolated more than 100 years ago and hydroxylamine is known to be an intermediate. The enzymatic steps involving hydroxylamine conversion to nitrite are still under discussion. For a long time it was assumed that hydroxylamine was directly converted to nitrite by a hydroxylamine oxidoreductase. Recent enzymatic evidences suggest that the actual product of hydroxylamine conversion is NO and a third, yet unknown, enzyme further converts NO to nitrite. More recently, ammonium oxidizing archaea and complete ammonium oxidizing bacteria were isolated and identified. Still the central nitrogen metabolism of these microorganisms presents to researchers the same puzzle: how hydroxylamine is transformed to nitrite. Nitrogen losses in the form of NO and N2O have been identified in all three types of aerobic ammonium oxidizing microorganisms and hydroxylamine is known to play a significant role in the formation. Yet, the pathways and the factors promoting the greenhouse gas emissions are to be fully characterized. Hydroxylamine also plays a yet poorly understood role on anaerobic ammonium oxidizing bacteria and is known to inhibit nitrite oxidizing bacteria. In this review, the role of this elusive intermediate in the metabolism of different key players of the nitrogen cycle is discussed, as well as the putative importance of hydroxylamine as a key nitrogen metabolite for microbial interactions within microbial communities and engineered systems. Overall, for the first time putting together the acquired knowledge about hydroxylamine and the nitrogen cycle over the years in a review, setting potential hypothesis and highlighting possible next steps for research.@en

Hydroxylamine is a nitrogen intermediate of ammonium oxidizing bacteria (AOB) that can transiently accumulate during nitrification. The impact of hydroxylamine on aerobic ammonium oxidations is still obscure. In the present study the short and long term impact of hydroxylamine on partial nitritation granular sludge was investigated. Dissolved oxygen was the governing factor determining the hydroxylamine impact in short term studies with continuous hydroxylamine feeding. Continuous short term hydroxylamine feeding together with low dissolved oxygen resulted in higher hydroxylamine accumulation, higher N2O production and decreased or maintained ammonium consumption. Instead, high dissolved oxygen reduced hydroxylamine accumulation and N2O production and increased ammonium consumption. Long term continuous hydroxylamine feeding reduced ammonium consumption rate while the constant nitrite production rate indicated that dosed hydroxylamine was mainly transformed to nitrite. This indicates that hydroxylamine was preferred over ammonium as substrate. Nitrosomonas sp. was shown to be predominant during continuous hydroxylamine feeding while the side community shifted.@en

Partial nitritation was stably achieved in a bench-scale airlift reactor (1.5L) containing granular sludge. Continuous operation at 20 °C treating low-strength synthetic wastewater (50 mg N-NH4 +/L and no COD) achieved nitrogen loading rates of 0.8 g N-NH4 +/(L·d) during partial nitritation. The switch between nitrite-oxidizing bacteria (NOB) repression and NOB proliferation was observed when ammonium concentrations in the reactor were below 2–5 mg N-NH4 +/L for DO concentrations lower than 4 mg O2/L at 20 °C. Nitrospira spp. were detected to be the dominant NOB population during the entire reactor operation, whereas Nitrobacter spp. were found to be increasing in numbers over time. Stratification of the granule structure, with ammonia-oxidizing bacteria (AOB) occupying the outer shell, was found to be highly important in the repression of NOB in the long term. The pH gradient in the granule, containing a pH difference of ca. 0.4 between the granule surface and the granule centre, creates a decreasing gradient of ammonia towards the centre of the granule. Higher residual ammonium concentration enhances the ammonium oxidation rate of those cells located further away from the granule surface, where the competition for oxygen between AOB and NOB is more important, and it contributes to the stratification of both populations in the biofilm.@en

A three-dimensional (3-D) diffusion-reaction model was used to assess the effects of nitrifiers growing in cell clusters on the apparent oxygen half-saturation coefficients in activated sludge flocs. The model allows conciliation of seemingly contradictory reports by several research groups. Although intrinsic half-saturation coefficients (i.e., not affected by diffusion) show a better affinity for oxygen for ammonia oxidizing (AOB) than for nitrite oxidizing bacteria (NOB) (KO,AOB <KO,NOB), measurements in flocs often produced reversed apparent values (KO,AOB,app > KO,NOB,app), which can now be explained by the 3-D model with AOB and NOB microcolonies. This effect cannot be described with a conventional 1-D homogeneous model because the reversion of the AOB/NOB apparent KO is caused by the high biomass density and resulting concentration gradients inside the microcolonies. Two main factors explain the reversion of the half-saturation coefficients: the difference in oxygen yields (for NOB lower than for AOB) and the difference in colony size (NOB colonies are smaller than those of AOB). The strongest increase in the apparent half-saturation coefficients is linked to the colony size, rather than to the floc size. For high-density microbial aggregates (i.e., granular sludge), the need for a stratified population (AOB outer shell, NOB inner layers) was revealed in order to outcompete NOB. This study stresses the need for a more detailed description of the biomass distribution in activated sludge, granular sludge and biofilm reactors when elucidating the mechanisms for NOB repression.@en

The enrichment of r-strategist ammonium oxidizing bacteria (r-AOB) to enhance nitritation and effective nitrogen removal was studied. A lab-scale three-compartment reactor system removing nitrogen from synthetic wastewater containing ammonium and COD (NH4 +:COD ratio at ca. 1:8) was used. A stable nitritation-denitritation process was achieved. The first compartment was anoxic for denitrification and the last two compartments were aerobic to oxidize ammonium into nitrite (nitritation). Recirculation between last and first compartment allowed for effective denitrification. Nitritation was achieved by enhancing the growth of fast growing r-AOB and setting a high NH4 + concentrations in the first of the two aerobic compartments (named as r-AOB selector). The sludge was wasted adaptively to maintain NH4 + concentration between 10 and 20 mg N/L in the r-AOB selector. The adaptive sludge wastage resulted in a short sludge retention time (SRT) of 4.2 days that led to full nitritation within 100 days of operation. Kinetic parameter estimation indicated that the maximum specific growth rate for AOB increased from 0.39 to 1.45 d−1, while the NH4 + half saturation constant increased from 0.51 to 5.23 mg N/L, indicating the transition from K-AOB (slower growing K-strategist AOB) to r-AOB. Mathematical simulation indicated that the SRT and NH4 + concentration were the main factors determining the outcome of nitrifying species competition.@en

The enrichment of r-strategist ammonium oxidizing bacteria (r-AOB) to enhance nitritation and effective nitrogen removal was studied. A lab-scale three-compartment reactor system removing nitrogen from synthetic wastewater containing ammonium and COD (NH4 +:COD ratio at ca. 1:8) was used. A stable nitritation-denitritation process was achieved. The first compartment was anoxic for denitrification and the last two compartments were aerobic to oxidize ammonium into nitrite (nitritation). Recirculation between last and first compartment allowed for effective denitrification. Nitritation was achieved by enhancing the growth of fast growing r-AOB and setting a high NH4 + concentrations in the first of the two aerobic compartments (named as r-AOB selector). The sludge was wasted adaptively to maintain NH4 + concentration between 10 and 20 mg N/L in the r-AOB selector. The adaptive sludge wastage resulted in a short sludge retention time (SRT) of 4.2 days that led to full nitritation within 100 days of operation. Kinetic parameter estimation indicated that the maximum specific growth rate for AOB increased from 0.39 to 1.45 d−1, while the NH4 + half saturation constant increased from 0.51 to 5.23 mg N/L, indicating the transition from K-AOB (slower growing K-strategist AOB) to r-AOB. Mathematical simulation indicated that the SRT and NH4 + concentration were the main factors determining the outcome of nitrifying species competition.@en