Ammonia-oxidizing bacteria (AOB) can use oxygen and nitrite as electron acceptors. Nitrite reduction by Nitrosomonas is observed under three conditions: (i) hydrogen-dependent denitrification, (ii) anoxic ammonia oxidation with nitrogen dioxide (NO2) and (iii) NOx-induced aerobic ammonia oxidation. NOx molecules play an important role in the conversion of ammonia and nitrite by AOB. Absence of nitric oxide (NO), which is generally detectable during ammonia oxidation, severely impairs ammonia oxidation by AOB. The lag phase of recovery of aerobic ammonia oxidation was significantly reduced by NO2 addition. Acetylene inhibition tests showed that NO2-dependent and oxygen-dependent ammonia oxidation can be distinguished. Addition of NOx increased specific activity of ammonia oxidation, growth rate and denitrification capacity. Together, these findings resulted in a hypothetical model on the role of NOx in ammonia oxidation: the NOx cycle.

Introduction

Ammonia-oxidizing bacteria (AOB) have been described as obligatory aerobic chemolithoautotrophic organisms for over 100 years. The only known pathway to gain energy was assumed to be aerobic oxidation of ammonia to nitrite. However, the appearance of aerobic nitrifiers in anoxic environments [1,2] indicated that AOB are more versatile than previously assumed. It was discovered that Nitrosomonas eutropha could also use nitrite as electron acceptor, which was observed under three conditions: (i) hydrogen-dependent denitrification, (ii) anoxic ammonia oxidation with NO2 [3] and (iii) NOx-induced aerobic ammonia oxidation. Nitrosospira spp. are also capable of nitrite reduction, suggesting that denitrification could be a universal trait in the β-proteobacterial AOB [4,5].

The pathways of aerobic and anoxic, NO2-dependent, ammonia oxidation are summarized in 1. The main products of ammonia oxidation are nitrite under oxic conditions and dinitrogen gas (N2), nitrite and NO under anoxic conditions. Both aerobic and anoxic ammonia oxidation to hydroxylamine is initiated by the enzyme AMO (ammonia mono-oxygenase). Ammonia, oxygen and NO2 are the substrates for this enzyme. Comparison of experiments at 25 and 4°C indicated that N2O4, and not NO2, is the actual molecule that is used [6]. The free energy change (ΔG°′) indicated that anoxic ammonia oxidation [−140 kJ·mol−1; eqn (2)] is slightly more exergonic than oxic ammonia oxidation [−120 kJ·mol−1; eqn (1)]:

 
formula
(1)

The hydroxylamine is in both cases further oxidized to nitrite by hydroxylamine oxidoreductase:

 
formula
(3)

Under anoxic conditions, the nitrite produced is partly used as electron acceptor, leading to the formation of N2 [3]:

 
formula
(4)

Under oxic conditions, with low NOx concentrations present, N. eutropha also uses nitrite as an additional electron acceptor, producing N2 and traces of nitrous oxide (N2O) [7].

Anoxic (A) and aerobic (B) ammonia oxidation of Nitrosomonas

Scheme 1
Anoxic (A) and aerobic (B) ammonia oxidation of Nitrosomonas

Under anoxic conditions, nitrite is the only electron acceptor available, leading to a high N loss via the denitrification pathway. Under oxic conditions, most electrons are transferred to oxygen and a minor share of the electrons is discharged via denitrification. Adapted from [12] with permission. © 2004 Horizon Scientific Press.

Scheme 1
Anoxic (A) and aerobic (B) ammonia oxidation of Nitrosomonas

Under anoxic conditions, nitrite is the only electron acceptor available, leading to a high N loss via the denitrification pathway. Under oxic conditions, most electrons are transferred to oxygen and a minor share of the electrons is discharged via denitrification. Adapted from [12] with permission. © 2004 Horizon Scientific Press.

In the absence of both dissolved oxygen and NO2, N. eutropha and N. europaea are capable of nitrite reduction with molecular hydrogen or acetate as electron donor [8]. The enzymes involved in the denitrification pathway in N. europaea seem to be regulated by a new type of protein [9].

Significance of NOx in aerobic ammonia oxidation

NOx plays an important role in the aerobic metabolism of nitrifying micro-organisms also when not added artificially. Release of NO during ammonia oxidation is well known, but was previously interpreted as formation of a by-product of nitrification, denitrification or chemodenitrification without greater significance for the metabolism of ammonia oxidizers. N. eutropha in aerobic laboratory-scale cultures was inhibited when gaseous NO was removed from the cultures, by means of intensive aeration, chemical binding with 2,3-dimercapto-1-propanesulphonic acid or removal by Pseudomonas PS 88 [10]. Nitrification in these cultures only started again when NO was added.

NOx seems to be necessary for the start-up of ammonia oxidation in N. europaea. The lag phase during recovery of aerobic ammonia oxidation, after growth on hydrogen and nitrite, was significantly reduced when NO2 was added [11]. Simultaneously, the arrangement of intracytoplasmic membranes changed from circular to flattened vesicles, the protein pattern revealed an increase in the concentrations of 27 and 30 kDa polypeptides, and the cytochrome c content increased significantly. When no NO2 was added, up to 10 p.p.m. NO was detected in the headspace of the cultures. This indicates that the cells generate NO if external NOx is not available.

Using a continuous aerobic laboratory-scale fermenter with biomass retention, it was shown that NO and NO2 have a stimulating effect on pure cultures of N. eutropha. Compared with cultures grown without these externally added NOx, there was an increase in specific activity of ammonia oxidation, increased growth rate and increased denitrification capacity. A major part of the ammonia was denitrified to N2, in the presence of low NO2 concentrations. The denitrification in the same system, but supplied with NO gas instead of NO2, was significantly lower [7]. It was hypothesized that, in the presence of NO, AOB elevate their denitrification capacity to compensate for reduced respiration activity, caused by inhibition of cytochrome oxidases by NO [12].

Significance of NOx in anoxic ammonia oxidation

Under anoxic conditions, between 40 and 60% of the produced nitrite is denitrified to N2. The specific ammonia oxidation activity is approx. 10-fold lower compared with aerobic conditions due to the fact that only low amounts of NO2 can be supplemented to the Nitrosomonas culture, as high levels are inhibitory [3].

Metabolic activities of Nitrosomonas europaea wild-type, and NirK- and NorB-deficient mutants

Three different N. europaea strains – wild-type, nitrite reductase (NirK)-deficient and nitric oxide reductase (NorB)-deficient strains – were characterized in chemostat cell cultures, and the effect of NO on metabolic activities was evaluated [13]. All strains revealed similar aerobic ammonia oxidation activities, but the growth rates and yields of the knockout mutants were significantly reduced. N2 was the main gaseous product of the wild-type, produced via its denitrification activity. The mutants were unable to reduce nitrite to N2, but excreted more hydroxylamine, leading to the formation of almost equal amounts of NO, N2O and N2 by chemical autoxidation and chemodenitrification of hydroxylamine. Under anoxic conditions N. europaea wild-type gains energy for growth via NO2-dependent ammonia oxidation or hydrogen-dependent denitrification using nitrite as electron acceptor. The mutant strains were restricted to NO and/or N2O as electron acceptor and consequently their growth rates and yields were much lower compared with the wild-type. These findings showed that the denitrification pathway is important for growth of AOB.

Discrimination between aerobic NO2-dependent and O2-dependent ammonia oxidation

Batch incubation experiments with and without the addition of acetylene showed that NO2-dependent and oxygen-dependent ammonia oxidation can be distinguished [14]. Ammonia oxidation by N. eutropha with NO2 as oxidant is not inhibited by acetylene, while oxygen-dependent ammonia oxidation is inhibited. The labelling reaction of a 27 kDa polypeptide with [14C]acetylene did not occur during anoxic NO2-dependent ammonia oxidation. When oxygen was added, the labelling of this polypeptide started immediately.

Nitrogen removal by co-culture of Candidatus ‘Brocadia anammoxidans’ and N. eutropha

The influence of NOx was tested on the combination of aerobic and anaerobic AOB [15]. B. anammoxidans converts ammonia and nitrite into N2 and nitrate. In the presence of NO2, the specific ammonia oxidation activity of B. anammoxidans increased, and Nitrosomonas-like micro-organisms recovered an NO2-dependent anoxic ammonia oxidation activity. The anammox bacterium was not inhibited by NO concentrations up to 600 p.p.m. and NO2 concentrations up to 100 p.p.m. Addition of NO2 to a mixed population of B. anammoxidans and Nitrosomonas induced simultaneous specific anaerobic ammonia oxidation activities of up to 5.5 mmol of NH4+·(g of protein)−1·h−1 by B. anammoxidans and up to 1.5 mmol of NH4+·(g of protein)−1·h−1 by Nitrosomonas. The stoichiometry of the converted N compounds (NO2/NH3 ratio) and the microbial community structure were strongly influenced by NO2. The combined activity of B. anammoxidans and Nitrosomonas-like ammonia oxidizers might be of relevance in natural environments and man-made ecosystems.

Effect of NO on biofilm formation

At a NO concentration of more than 30 p.p.m., biofilm formation by N. europaea was induced in a continuous aerobic laboratory-scale fermenter [16]. NO concentrations below 5 p.p.m. led to a reversal of the biofilm formation, and the numbers of motile and planktonic (motile-planktonic) cells increased. In a proteomics approach, the six proteins down-regulated by NO in N. europaea were identified as flagellar and flagellar assembly proteins.

Hypothetical model of the NOx cycle during ammonium oxidation

In order to explain the influence of NOx on ammonia oxidation, a hypothetical model (2) was developed [14]. The model summarizes the effects of NOx on ammonia oxidation, as presented in this paper. N2O4 is the oxidizing agent for ammonia oxidation, producing hydroxylamine and NO. Under oxic conditions, NO is reoxidized to NO2, again providing the AMO with the oxidizing agent (NOx cycle). Since detectable NOx concentrations were small, NOx seems to cycle in the cell (possibly enzyme-bound) and, therefore, the total amount of NOx per cell is expected to be low. The addition of acetylene leads to an inhibition of aerobic ammonia oxidation, if NO2 is not present. The cells restart ammonia oxidation when NO2 is added, and NO is produced in stoichiometric amounts (ratio of NO2 consumption to NO production is approx. 1:1).

NOx cycle

Scheme 2
NOx cycle

Hypothetical model of ammonia oxidation by N. eutropha. Reproduced from [14] with permission. © 2001 Society for General Microbiology.

Scheme 2
NOx cycle

Hypothetical model of ammonia oxidation by N. eutropha. Reproduced from [14] with permission. © 2001 Society for General Microbiology.

Conclusion

NOx plays an important role in the metabolism of AOB. NOx enhances recovery of aerobic ammonia oxidation, growth of AOB and denitrification activity. A hypothetical model was developed to explain the influence of NOx on ammonia oxidation. The exact mechanism behind the stimulating effect of NOx still needs to be elucidated. The ecophysiological role of NOx in nitrogen conversion also needs further clarification.

The 11th Nitrogen Cycle Meeting 2005: Independent Meeting held at Estación Experimental del Zaidín, Granada, Spain, 15–17 September 2005. Organized and Edited by E.J. Bedmar (Granada, Spain), M.J. Delgado (Granada, Spain) and C. Moreno-Vivián (Córdoba, Spain).

Abbreviations

     
  • AMO

    ammonia mono-oxygenase

  •  
  • AOB

    ammonia-oxidizing bacteria

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