Currently available microbiological techniques are not designed to deal with very slowly growing microorganisms. The enrichment and study of such organisms demands a novel experimental approach. In the present investigation, the sequencing batch reactor (SBR) was applied and optimized for the enrichment and quantitative study of a very slowly growing microbial community which oxidizes ammonium anaerobically. The SBR was shown to be a powerful experimental set-up with the following strong points: (1) efficient biomass retention, (2) a homogeneous distribution of substrates, products and biomass aggregates over the reactor, (3) reliable operation for more than 1 year, and (4) stable conditions under substrate-limiting conditions. Together, these points made possible for the first time the determination of several important physiological parameters such as the biomass yield (0.066 ± 0.01 C-mol/mol ammonium), the maximum specific ammonium consumption rate (45 ± 5 nmol/mg protein/min) and the maximum specific growth rate (0.0027 · h−1, doubling time 11 days). In addition, the persisting stable and strongly selective conditions of the SBR led to a high degree of enrichment (74% of the desired microorganism). This study has demonstrated that the SBR is a powerful tool compared to other techniques used in the past. We suggest that the SBR could be used for the enrichment and quantitative study of a large number of slowly growing microorganisms that are currently out of reach for microbiological research.

The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms

Nitrifier denitrification is the pathway of nitrification in which ammonia (NH 3 ) is oxidized to nitrite (NO 2 − ) followed by the reduction of NO 2 − to nitric oxide (NO), nitrous oxide (N 2 O) and molecular nitrogen (N 2 ). The transformations are carried out by autotrophic nitrifiers. Thus, nitrifier denitrification differs from coupled nitrification–denitrification, where denitrifiers reduce NO 2 − or nitrate (NO 3 − ) that was produced by nitrifiers. Nitrifier denitrification contributes to the development of the greenhouse gas N 2 O and also causes losses of fertilizer nitrogen in agricultural soils. In this review article, present knowledge about nitrifier denitrification is summarized in order to give an exact definition, to spread awareness of its pathway and controlling factors and to identify areas of research needed to improve global N 2 O budgets. Due to experimental difficulties and a lack of awareness of nitrifier denitrification, not much is known about this mechanism of N 2 O production. The few measurements carried out so far attribute up to 30% of the total N 2 O production to nitrifier denitrification. Low oxygen conditions coupled with low organic carbon contents of soils favour this pathway as might low pH. As nitrifier denitrification can lead to substantial N 2 O emissions, there is a need to quantify this pathway in different soils under different conditions. New insights attained through quantification experiments should be used in the improvement of computer models to define sets of conditions that show where and when nitrifier denitrification is a significant source of N 2 O. This may subsequently render the development of guidelines for low-emission farming practices necessary.

Role of nitrifier denitrification in the production of nitrous oxide

▪ Abstract The eutrophication of many ecosystems in recent decades has led to an increased interest in the ecology of nitrogen transformation. Chemolitho-autotrophic ammonia-oxidizing bacteria are responsible for the rate-limiting step of nitrification in a wide variety of environments, making them important in the global cycling of nitrogen. These organisms are unique in their ability to use the conversion of ammonia to nitrite as their sole energy source. Because of the importance of this functional group of bacteria, understanding of their ecology and physiology has become a subject of intense research over recent years. The monophyletic nature of these bacteria in terrestrial environments has facilitated molecular biological approaches in studying their ecology, and progress in this field has been rapid. The ammonia-oxidizing bacteria of the β-subclass Proteobacteria have become somewhat of a model system within molecular microbial ecology, and this chapter reviews recent progress in our knowledge of ...

Ammonia-Oxidizing Bacteria: A Model for Molecular Microbial Ecology

Nitrous oxide emission during wastewater treatment.

From recent research it has become clear that at least two different possibilities for anaerobic ammonium oxidation exist in nature. 'Aerobic' ammonium oxidizers like Nitrosomonas eutropha were observed to reduce nitrite or nitrogen dioxide with hydroxylamine or ammonium as electron donor under anoxic conditions. The maximum rate for anaerobic ammonium oxidation was about 2 nmol NH4+ min-1 (mg protein)-1 using nitrogen dioxide as electron acceptor. This reaction, which may involve NO as an intermediate, is thought to generate energy sufficient for survival under anoxic conditions, but not for growth. A novel obligately anaerobic ammonium oxidation (Anammox) process was recently discovered in a denitrifying pilot plant reactor. From this system, a highly enriched microbial community with one dominating peculiar autotrophic organism was obtained. With nitrite as electron acceptor a maximum specific oxidation rate of 55 nmol NH4+ min-1 (mg protein)-1 was determined. Although this reaction is 25-fold faster than in Nitrosomonas, it allowed growth at a rate of only 0.003 h-1 (doubling time 11 days). 15N labeling studies showed that hydroxylamine and hydrazine were important intermediates in this new process. A novel type of hydroxylamine oxidoreductase containing an unusual P468 cytochrome has been purified from the Anammox culture. Microsensor studies have shown that at the oxic/anoxic interface of many ecosystems nitrite and ammonia occur in the absence of oxygen. In addition, the number of reports on unaccounted high nitrogen losses in wastewater treatment is gradually increasing, indicating that anaerobic ammonium oxidation may be more widespread than previously assumed. The recently developed nitrification systems in which oxidation of nitrite to nitrate is prevented form an ideal partner for the Anammox process. The combination of these partial nitrification and Anammox processes remains a challenge for future application in the removal of ammonium from wastewater with high ammonium concentrations.

The anaerobic oxidation of ammonium

Cells of the obligately lithotrophic species Nitrosomonas europaea and Nitrosomonas eutropha were able to nitrify and denitrify at the same time when grown under oxygen limitation. In addition to oxygen, nitrite was used as an electron acceptor. The simultaneous nitrification and denitrification resulted in significant formation of the gaseous N-compounds nitrous oxide and dinitrogen, causing significant nitrogen loss. In mixed cultures of N. europaea and various chemoorganotrophic bacteria, the nitrogen loss was strongly influenced by the partners growing under oxygen limitation. Under anoxic conditions, pure cultures of N. eutropha were able to denitrify with molecular hydrogen as electron donor and nitrite as the only electron acceptor in a sulfide-reduced complex medium. The increase of cell numbers was directly coupled to nitrite reduction. Nitrous oxide and dinitrogen were the only detectable end products. In pure cultures of N. eutropha and mixed cultures of N. eutropha and Enterobacter aerogenes, ammonium and nitrite disappeared slowly at a molar ratio of about one when oxygen was absent. However, under these conditions cell growth was not measurable.

Nitrogen loss caused by denitrifying Nitrosomonas cells using ammonium or hydrogen as electron donors and nitrite as electron acceptor

Chemolithoautotrophically growing cells of Nitrosomonas europaea quantitatively oxidized ammonia to nitrite under aerobic conditions with no loss of inorganic nitrogen. Significant inorganic nitrogen losses occurred when cells were growing mixotrophically with ammonium, pyruvate, yeast extract and peptone. Under oxygen limitation the nitrogen losses were even higher. In the absence of oxygen pyruvate was metabolized slowly while nitrite was consumed concomitantly. Nitrogen losses were due to the production of nitric oxide and nitrous oxide. In mixed cultures of Nitrosomonas and Nitrobacter, strong inhibition of nitrite oxidation was reproducibly measured. NO and ammonium were not inhibitory to Nitrobacter. First evidence is given that hydroxylamine, the intermediate of the Nitrosomonas monooxygenase-reaction, is formed. 0.2 to 1.7 μM NH2OH were produced by mixotrophically growing cells of Nitrosomonas and Nitrosovibrio. Hydroxylamine was both a selective inhibitory agent to Nitrobacter cells and a strong reductant which reduced nitrite to NO and N2O. It is discussed whether chemodenitrification or denitrification is the most abundant process for NO and N2O production of Nitrosomonas.

Ralf Stüven

Papers

Nitrogen loss caused by denitrifying Nitrosomonas cells using ammonium or hydrogen as electron donors and nitrite as electron acceptor

The impact of organic matter on nitric oxide formation by Nitrosomonas europaea

Nitrification and denitrification as a source for NO and NO2 production in high-strength wastewater.

Nitrification and Denitrification‐Microbial Fundamentals and Consequences for Application

Ein neues Verfahren zur Entfernung von Ammonium‐Stickstoff aus Abwasser