Metabolic modeling of denitrification in Agrobacterium tumefaciens: a tool to study inhibiting and activating compounds for the denitrification pathway.

A metabolic network model for facultative denitrification was developed based on experimental data obtained with Agrobacterium tumefaciens. The model includes kinetic regulation at the enzyme level and transcription regulation at the enzyme synthesis level. The objective of this work was to study the key factors regulating the metabolic response of the denitrification pathway to transition from oxic to anoxic respiration and to find parameter values for the biological processes that were modeled. The metabolic model was used to test hypotheses that were formulated based on the experimental results and offers a structured look on the processes that occur in the cell during transition in respiration. The main phenomena that were modeled are the inhibition of the cytochrome c oxidase by nitric oxide (NO) and the (indirect) inhibition of oxygen on the denitrification enzymes. The activation of transcription of nitrite reductase and NO reductase by their respective substrates were hypothesized. The general assumption that nitrite and NO reduction are controlled interdependently to prevent NO accumulation does not hold for A. tumefaciens. The metabolic network model was demonstrated to be a useful tool for unraveling the different factors involved in the complex response of A. tumefaciens to highly dynamic environmental conditions.

Analysis of ammonia-oxidizing bacteria dominating in lab-scale bioreactors with high ammonium bicarbonate loading.

The ammonia-oxidizing bacterial community (AOB) was investigated in two types of laboratory-scale bioreactors performing partial oxidation of ammonia to nitrite or nitrate at high (80 mM) to extremely high (428 mM) concentrations of ammonium bicarbonate. At all conditions, the dominant AOB was affiliated to the Nitrosomonas europaea lineage as was determined by fluorescence in situ hybridization and polymerase chain reaction in combination with denaturing gradient gel electrophoresis. Molecular analysis of the mixed populations, based on the 16S rRNA and cbbL genes, demonstrated the presence of two different phylotypes of Nitrosomonas, while microbiological analysis produced a single phylotype, represented by three different morphotypes. One of the most striking features of the AOB populations encountered in the bioreactors was the domination of highly aggregated obligate microaerophilic Nitrosomonas, with unusual cellular and colony morphology, commonly observed in nitrifying bioreactors but rarely investigated by cultural methods. The latter is probably not an adaptation to stressful conditions created by high ammonia or nitrite concentrations, but oxygen seems to be a stressful factor in these bioreactors.

Reduced iron induced nitric oxide and nitrous oxide emission.

Formation of the greenhouse gas nitrous oxide in water treatment systems is predominantly studied as a biological phenomenon. There are indications that also chemical processes contribute to these emissions. Here we studied the formation of nitric oxide (NO) and nitrous oxide (N(2)O) due to chemical nitrite reduction by ferrous iron (Fe(II)). Reduction of nitrite and NO coupled to Fe(II) oxidation was studied in laboratory-scale chemical experiments at different pH, nitrite and iron concentrations. The continuous measurement of both NO and N(2)O emission showed that nitrite reduction and NO reduction have different kinetics. Nitrite reduction shows a linear dependency on the nitrite concentration, implying first order kinetics in nitrite. The nitrite reduction seems to be an equilibrium based reaction, leading to a constant NO concentration in the liquid. The NO reduction rate is suggested to be most dependent on reactive surface availability and the sorption of Fe(II) to the reactive surface. The importance of emission of NO and N(2)O coupled to iron oxidation is exemplified by iron reduction experiments and several examples of environments where this pathway can play a role.

Effect of nitric oxide on anammox bacteria.

The effects of nitrogen oxides on anammox bacteria are not well known. Therefore, anammox bacteria were exposed to 3,500 ppm nitric oxide (NO) in the gas phase. The anammox bacteria were not inhibited by the high NO concentration but rather used it to oxidize additional ammonium to dinitrogen gas under conditions relevant to wastewater treatment.

Mechanisms and specific directionality of autotrophic nitrous oxide and nitric oxide generation during transient anoxia.

The overall goal of this study was to determine the molecular and metabolic responses of chemostat cultures of model nitrifying bacteria to imposition of and recovery from transient anoxic conditions. Based on the study, a specific directionality in nitrous oxide (N(2)O) and nitric oxide (NO) production was demonstrated. N(2)O production was only observed during recovery to aerobic conditions after a period of anoxia and correlated positively with the degree of ammonia accumulation during anoxia. NO, on the other hand, was emitted mainly under anoxia. The production of NO was linked to a major imbalance in the expression of the nitrite reductase gene, which was overexpressed during transient anoxia. In contrast, genes coding for ammonia and hydroxylamine oxidation and nitric oxide reduction were generally under-expressed during transient anoxia. These results are different from the observed parallel expression and activity of nitrite and nitric oxide reductase in heterotrophic bacteria subjected to transient oxygen cycling. Unlike NO, the production of N(2)O could not be solely correlated to gene expression patterns and likely involved responses at the enzyme activity or metabolic levels. Based on experimental data, the propensity of the nitrifying cultures for N(2)O production is related to a shift in their metabolism from a low specific activity (q < q(max)) toward the maximum specific activity (q(max)).

Nitrous oxide emission during wastewater treatment.

Nitrous oxide (N(2)O), a potent greenhouse gas, can be emitted during wastewater treatment, significantly contributing to the greenhouse gas footprint. Measurements at lab-scale and full-scale wastewater treatment plants (WWTPs) have demonstrated that N(2)O can be emitted in substantial amounts during nitrogen removal in WWTPs, however, a large variation in reported emission values exists. Analysis of literature data enabled the identification of the most important operational parameters leading to N(2)O emission in WWTPs: (i) low dissolved oxygen concentration in the nitrification and denitrification stages, (ii) increased nitrite concentrations in both nitrification and denitrification stages, and (iii) low COD/N ratio in the denitrification stage. From the literature it remains unclear whether nitrifying or denitrifying microorganisms are the main source of N(2)O emissions. Operational strategies to prevent N(2)O emission from WWTPs are discussed and areas in which further research is urgently required are identified.

Modelling nitrite in wastewater treatment systems: a discussion of different modelling concepts.

Originally presented at the 1st IWA/WEF Wastewater Treatment Modelling Seminar (WWTmod 2008), this contribution has been updated to also include the valuable feedback that was received during the Modelling Seminar. This paper addresses a number of basic issues concerning the modelling of nitrite in key processes involved in biological wastewater water treatment. To this end, we review different model concepts (together with model structures and corresponding parameter sets) proposed for processes such as two-step nitrification/denitrification, anaerobic ammonium oxidation and phosphorus uptake processes. After critically discussing these models with respect to their assumptions and parameter sets, common points of agreement as well as disagreement were elucidated. From this discussion a general picture of the state-of-the-art in the modelling of nitrite is provided. Taking this into account, a number of recommendations are provided to focus further research and development on nitrite modelling in biological wastewater treatment.

Physiological and phylogenetic study of an ammonium-oxidizing culture at high nitrite concentrations.

Oxidation of high-strength ammonium wastewater can lead to exceptionally high nitrite concentrations; therefore, the effect of high nitrite concentration (> 400 mM) was studied using an ammonium-oxidizing enrichment culture in a batch reactor. Ammonium was fed to the reactor in portions of 40-150 mM until ammonium oxidation rates decreased and finally stopped. Activity was restored by replacing half of the medium, while biomass was retained by a membrane. The ammonium-oxidizing population obtained was able to oxidize ammonium at nitrite concentrations of up to 500 mM. The maximum specific oxidation activity of the culture in batch test was about 0.040 mmol O(2)g(-1)proteinmin(-1) and the K(s) value was 1.5 mM ammonium. In these tests, half of the maximum oxidation activity was still present at a concentration of 600 mM nitrite and approximately 10% residual activity could still be measured at 1200 mM nitrite (pH 7.4), or as a free nitrous acid (FNA) concentration of 6.6 mg l(-1). Additional experiments showed that the inhibition was caused by nitrite and not by the high sodium chloride concentration of the medium. The added ammonium was mainly converted into nitrite and no nitrite oxidation was observed. In addition, gaseous nitrogen compounds were detected and mass balance calculations revealed a nitrogen loss of approximately 20% using this system. Phylogenetic analyses of 16S rRNA and ammonium monooxygenase (amoA) genes of the obtained enrichment culture showed that ammonium-oxidizing bacteria of the Nitrosomonas europaea/Nitrosococcus mobilis cluster dominated the two clone libraries. Approximately 25% of the 16S rRNA clones showed a similarity of 92% to Deinococcus-like organisms. Specific fluorescence in situ hybridization (FISH) probes confirmed that these microbes comprised 10-20% of the microbial community in the enrichment. The Deinococcus-like organisms were located around the Nitrosomonas clusters, but their role in the community is currently unresolved.

Effect of dynamic process conditions on nitrogen oxides emission from a nitrifying culture.

Nitric oxide (NO) and nitrous oxide (N2O) emissions from nitrifying ecosystems are a serious threat to the environment. The factors influencing the emission and the responsible microorganisms and pathways were studied using a laboratory-scale nitrifying reactor system. The nitrifying culture was established at growth rates relevant to wastewater treatment plants (WWTPs). During stable ammonia oxidation, 0.03% of ammonium was emitted as NO and 2.8% was emitted as N2O. Although mixed cultures were used, clear responses in emission of ammonia oxidizing bacteria (AOB) could be detected and it was concluded that the denitrification pathway of AOB was the main source of the emissions. Emissions of nitrogen oxides in the system were strongly influenced by oxygen, nitrite, and ammonium concentrations. Steady state emission levels greatly underestimate the total emission, because changes in oxygen, nitrite, and ammonium concentrations induced a dramatic rise in NO and N2O emission. The data presented can be used as an indication for NO and N2O emission by AOB in plug-flow activated sludge systems, which is highly relevant because of the atmospheric impact and potential health risk of these compounds.

Dynamics of nitric oxide and nitrous oxide emission during full-scale reject water treatment.

Emission of NO and N2O from a full-scale two-reactor nitritation-anammox process was determined during a measurement campaign at the Dokhaven-Sluisjesdijk municipal WWTP (Rotterdam, NL). The NO and N2O levels in the off-gas responded to the aeration cycles and the aeration rate of the nitritation reactor, and to the nitrite and dissolved oxygen concentration. Due to the strong fluctuations in the NO and N2O levels in both the nitritation and the anammox reactor, only time-dependent measurements could yield a reliable estimate of the overall NO and N2O emissions. The NO emission from the nitritation reactor was 0.2% of the nitrogen load and the N2O emission was 1.7%. The NO emission from the anammox reactor was determined to be 0.003% of the nitrogen load and the N2O emission was 0.6%. Emission of NO2 could not be detected from the nitritation-anammox system. Denitrification by ammonia-oxidizing bacteria was considered to be the most probable cause of NO and N2O emission from the nitritation reactor. Since anammox bacteria have not been shown to produce N2O under physiological conditions, it is also suspected that ammonia-oxidizing bacteria contribute most to N2O production in the anammox reactor. The source of NO production in the anammox reactor can be either anammox bacteria or denitrification by heterotrophs or ammonia-oxidizing bacteria. Based on the results and previous work, it seems that a low dissolved oxygen or a high nitrite concentration are the most likely cause of elevated NO and N2O emission by ammonia-oxidizing bacteria. The emission was compared with measurements at other reject water technologies and with the main line of the Dokhaven-Sluisjesdijk WWTP. The N2O emission levels in the reject water treatment seem to be in the same range as for the main stream of activated sludge processes. Preliminary measurements of the N2O emission from a one-reactor nitritation-anammox system indicate that the emission is lower than in two-reactor systems.

Unraveling the source of nitric oxide emission during nitrification.

Nitric oxide production was measured during nitrification in a laboratory-scale bioreactor, operated at conditions relevant to municipal nitrifying wastewater treatment plants. This study aims to determine which type of microorganism and which metabolic pathway is responsible for nitric oxide emission during nitrification. Simulation studies were used to identify which pathway is the main source of nitric oxide emission, based on the following three hypothetical pathways for nitric oxide emission: (a) nitrification, (b) denitrification by ammonia-oxidizing bacteria with ammonium as electron donor, and (c) heterotrophic denitrification. The results of the study suggest that, in a nitrifying reactor treating wastewater containing solely ammonium and nutrients, denitrification by ammonia-oxidizing bacteria is the main nitric-oxide-producing pathway. During the experiments, 0.025% of the treated ammonium is emitted as nitric oxide, independent of the aeration rate imposed. Nitrite presence and oxygen limitation were found to increase the nitric oxide emission.