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Ammonia oxidation as the first step of nitrification constitutes a critical process in the global nitrogen cycle. However, fundamental knowledge of its key enzyme, the copper-dependent ammonia monooxygenase is lacking, in particular for the environmentally abundant ammonia oxidizing archaea (AOA). Here, the structure of the enzyme is investigated by blue-native gel electrophoresis and proteomics from native membrane complexes of two AOA. Beside the known AmoABC subunits and the earlier predicted AmoX, two new protein subunits, AmoY and AmoZ, were identified. They are unique to AOA, highly conserved and co-regulated, and their genes are linked to other AMO subunit genes in streamlined AOA genomes. Modelling and in gel cross-link approaches support an overall protomer structure similar to the distantly related bacterial particulate methane monooxygenase indicating that AmoY and AmoZ serve an important structural and functional role. These data open avenues for further structure-function studies of this ecologically important key nitrification complex.

To enable long-distance space travel, the development of a highly efficient and robust system to recover nutrients from waste streams is imperative. The inability of the current physicochemical-based environmental control and life support system (ECLSS) on the ISS to produce food in situ and to recover water and oxygen at high enough efficiencies results in the need for frequent resupply missions from Earth. Therefore, alternative strategies like biologically-based technologies called bioregenerative life support systems (BLSSs) are in development. These systems aim to combine biological and physicochemical processes, which enable in situ water, oxygen, and food production (through the highly efficient recovery of minerals from waste streams). Hence, minimalizing the need for external consumables. One of the BLSS initiatives is the European Space Agency’s (ESA) Micro-Ecological Life Support System Alternative (MELiSSA). It has been designed as a five-compartment bioengineered system able to produce fresh food and oxygen and to recycle water. As such, it could sustain the needs of a human crew for long-term space exploration missions. A prerequisite for the self-sufficient nature of MELiSSA is the highly efficient recovery of valuable minerals from waste streams. The produced nutrients can be used as a fertilizer for food production. In this review, we discuss the need to shift from the ECLSS to a BLSS, provide a summary of past and current BLSS programs and their unique approaches to nitrogen recovery and processing of urine waste streams. In addition, compartment III of the MELiSSA loop, which is responsible for nitrogen recovery, is reviewed in-depth. Finally, past, current, and future related ground and space demonstration and the space-related challenges for this technology are considered.

During long-term extra-terrestrial missions, food is limited and waste is generated. By recycling valuable nutrients from this waste via regenerative life support systems, food can be produced in space. Astronauts' urine can, for instance, be nitrified by micro-organisms into a liquid nitrate fertilizer for plant growth in space. Due to stringent conditions in space, microbial communities need to be be defined (gnotobiotic); therefore, synthetic rather than mixed microbial communities are preferred. For urine nitrification, synthetic communities face challenges, such as from salinity, ureolysis, and organics. In this study, a synthetic microbial community containing an AOB (Nitrosomonas europaea), NOB (Nitrobacter winogradskyi), and three ureolytic heterotrophs (Pseudomonas fluorescens, Acidovorax delafieldii, and Delftia acidovorans) was compiled and evaluated for these challenges. In reactor 1, salt adaptation of the ammonium-fed AOB and NOB co-culture was possible up to 45mScm-1, which resembled undiluted nitrified urine, while maintaining a 44±10mgNH4+-NL-1d-1 removal rate. In reactor 2, the nitrifiers and ureolytic heterotrophs were fed with urine and achieved a 15±6mg NO3--NL-1d-1 production rate for 1% and 10% synthetic and fresh real urine, respectively. Batch activity tests with this community using fresh real urine even reached 29±3mgNL-1d-1. Organics removal in the reactor (69±15%) should be optimized to generate a nitrate fertilizer for future space applications. ispartof: SYSTEMATIC AND APPLIED MICROBIOLOGY vol:42 issue:6 ispartof: location:Germany status: published

Nitrifier denitrification (i.e. nitrite reduction by ammonia oxidizers) is one of the biochemical pathways of nitrous oxide (N2O) production. It is increasingly suggested that this pathway may contribute substantially to N2O production in soil, the major source of this greenhouse gas. However, although monoculture studies recognize its potential, methodological drawbacks prohibit conclusive proof that nitrifier denitrification occurs in actual soils. Here we suggest and apply a new isotopic approach to identify its presence in soil. In incubation experiments with 12 soils, N2O production was studied using oxygen (O) and nitrogen (N) isotope tracing, accounting for O exchange. Microbial biomass C and N and phospholipid fatty acid (PLFA) patterns were analysed to explain potential differences in N2O production pathways. We found that in at least five of the soils nitrifier denitrification must have contributed to N2O production. Moreover, it may even have been responsible for all NH4+-derived N2O in most soils. In contrast, N2O as a by-product of ammonia oxidation contributed very little to total production. Microbial biomass C and N and PLFA-distinguished microbial community composition were not indicative of differences in N2O production pathways. Overall, we show that combined O and N isotope tracing may still provide a powerful tool to understand N2O production pathways, provided that O exchange is accounted for. We conclude that nitrifier denitrification can indeed occur in soils, and may in fact be responsible for the greater proportion of total nitrifier-induced N2O production.

We present a novel O-18-N-15-enrichment method for the distinction between nitrous oxide (N2O) from nitrification, nitrifier denitrification and denitrification based on a method with single- and double-N-15-labelled ammonium nitrate. We added a new treatment with O-18-labelled water to quantify N2O from nitrifier denitrification. The theory behind this is that ammonia oxidisers use oxygen (O-2) from soil air for the oxidation of ammonia (NH3) but use H2O for the oxidation of the resulting hydroxylamine (NH2OH) to nitrite (NO2-). Thus, N2O from nitrification would therefore be expected to reflect the O-18 signature of soil O-2, whereas the O-18 signature of N2O from nitrifier denitrification would reflect that of both soil O-2 and H2O. It was assumed that (a) there would be no preferential removal of O-18 or O-16 during nitrifier denitrification or denitrification, (b) the O-18 signature of the applied O-18-labelled water would remain constant over the experimental period, and (c) any O exchange between (H2O)-O-18 and NO3- would be negligible under the chosen experimental conditions. These assumptions were tested and validated for a silt loam soil at 50% water-filled pore space (WFPS) following application of 400 mg N kg(-1) dry soil. We compared the results of our new method with those of a conventional inhibition method using 0.02% v/v acetylene (C2H2) and 80% v/v O-2 in helium. Both the O-18-N-15-enrichment and inhibitor methods identified nitrifter denitrification to be a major source of N2O, accounting for 44 and 40%, respectively, of N2O production over 24 h. However, compared to our O-18-N-15-method, the inhibitor method overestimated the contribution from nitrification at the expense of denitrification, probably due to incomplete inhibition of nitrifier denitrification and denitrification by large concentrations Of 02 and a negative effect of C2H2 on denitrification. We consider our new O-18-N-15-enrichment method to be more reliable than the use of inhibitors; it enables the distinction between more soil sources of N2O than was previously possible and has provided the first direct evidence of the significance of nitrifier denitrification as a source of N2O in fertilised arable soil.

Wastes from animal production systems contribute as much as 30-50% to the global N2O emissions from agriculture, but relatively little attention has been given on improving the accuracy of the estimates and on developing mitigation options. This paper discusses trends and uncertainties in global N2O emission from animal waste and discusses possible mitigation strategies, on the basis of literature data and results of simple calculations. Total N2O emissions from animal production systems are estimated at 1.5 Tg. Dung and urine from grazing animals deposited in pastures (41%), indirect sources (27%), animal wastes in stables and storages (19%), application of animal wastes to land (10%) and burning of dung (3%) are the five sources distinguished. Most sensitive factors are N excretion per animal head, the emission factor for grazing animals and that for indirect emissions. Total N2O emissions are related to type and number of animals, N excretion per animal, and the management of animal wastes. Projections by FAO suggest that animal numbers will increase by 40% between 2000 and 2030. Mean N excretion per animal head will probably also increase. These trends combined suggest a strong increase in total N2O emission from animal production systems in the near future, which is opposite to the objectives of the Kyoto Protocol. Improving N use efficiency, combined with anaerobic digestion of animal wastes for bio fuel generation are the most feasible options for mitigation, but these options seem insufficient to reverse the trend of increasing N2O emission. In conclusion, animal production systems are a major and increasing source of N2O in agriculture. The uncertainties in the emission estimates are large, due to the many complexities involved and the lack of accurate data, especially about N excretion and the management of animal wastes in practice. Suggestions are made how to increase the accuracy of the emission estimates and to mitigate N2O emission from animal production systems.

Nitrifiers and denitrifiers are the main producers of the greenhouse gas nitrous oxide (N(2)O). Knowledge of the respective contributions of each of these microbial groups to N(2)O production is a prerequisite for the development of effective mitigation strategies for N(2)O. Often, the differentiation is made by the use of inhibitors. Measurements of the natural abundance of the stable isotopes of N and O in N(2)O have been suggested as an alternative for the often unreliable inhibition studies. Here, we tested the natural abundance incubation method developed by Tilsner et al.1 with soils from four European grasslands differing in long-term management practices. Emission rates of N(2)O and stable isotope natural abundance of N(2)O and mineral N were measured in four different soil incubations: a control with 60% water-filled pore space (WFPS), a treatment with 60% WFPS and added ammonium (NH(4) (+)) to support nitrifiers, a control with 80% WFPS and a treatment with 80% WFPS and added nitrate (NO(3) (-)) to support denitrifiers. Decreases in NH(4) (+) concentrations, linked with relative (15)N-enrichment of residual NH(4) (+) and production of (15)N-depleted NO(3) (-), showed that nitrification was the main process for mineral N conversions. The N(2)O production, however, was generally dominated by reduction processes, as indicated by the up to 20 times larger N(2)O production under conditions favouring denitrification than under conditions favouring nitrification. Interestingly, the N(2)O concentration in the incubation atmospheres often levelled off or even decreased, accompanied by increases in delta(15)N and delta(18)O values of N(2)O. This points to uptake and further reduction of N(2)O to N(2), even under conditions with small concentrations of N(2)O in the atmosphere. The measurements of the natural abundances of (15)N and (18)O proved to be a valuable integral part of the natural abundance incubation method. Without these measurements, nitrification would not have been identified as essential for mineral N conversions and N(2)O consumption could not have been detected.

We analysed the stable isotope composition of emitted N2O in a one-year field experiment (June 1998 to April 1999) in unfertilized controls, and after adding nitrogen by applying slurry or mineral N (calcium ammonium nitrate). Emitted N2O was analysed every 2¿4 weeks, with additional daily sampling for 10 days after each fertilizer application. In supplementary soil incubations, the isotopic composition of N2O was measured under defined conditions, favouring either denitrification or nitrification. Soil incubated for 48 h under conditions favouring nitrification emitted very little N2O (0.024 mol gdw ¿1) and still produced N2O from denitrification. Under denitrifying incubation conditions, much more N2O was formed (0.91 mol gdw ¿1 after 48 h). The isotope ratios of N2O emitted from denitrification stabilized at 15N = ¿40.8 ± 5.7 and 18O = 2.7 ± 6.3. In the field experiment, the N2O isotope data showed no clear seasonal trends or treatment effects. Annual means weighted by time and emission rate were 15N = ¿8.6 and 18O = 34.7 after slurry application, 15N = ¿4.6 and 18O = 24.0 after mineral fertilizer application and 15N = ¿6.4 and 18O = 35.6 in the control plots, respectively. So, in all treatments the emitted N2O was 15N-depleted compared to ambient air N2O (15N = 11.4 ± 11.6, 18O = 36.9 ± 10.7). Isotope analyses of the emitted N2O under field conditions per se allowed no unequivocal identification of the main N2O producing process. However, additional data on soil conditions and from laboratory experiments point to denitrification as the predominant N2O source. We concluded (1) that the isotope ratios of N2O emitted from the field soil were not only influenced by the source processes, but also by microbial reduction of N2O to N2 and (2) that N2O emission rates had to exceed 3.4 mol N2O m¿2 h¿1 to obtain reliable N2O isotope data

About 30 full-scale partial nitritation/anammox plants are established, treating mostly sewage sludge reject water, landfill leachate or food processing digestate. Although two-stage and one-stage processes each have their advantages, the one-stage configuration is mostly applied, termed here as oxygen-limited autotrophic nitrification/denitrification (OLAND), and is the focus of this review. The OLAND application domain is gradually expanding, with technical-scale plants on source-separated domestic wastewater, pre-treated manure and sewage, and liquors from organic waste bioenergy plants. A microbial resource management (MRM) OLAND framework was elaborated, showing how the OLAND engineer/operator (1: input) can design/steer the microbial community (2: biocatalyst) to obtain optimal functionality (3: output). In the physicochemical toolbox (1), design guidelines are provided, as well as advantages of different reactor technologies. Particularly the desirable aeration regime, feeding regime and shear forces are not clear yet. The development of OLAND trickling filters, membrane bioreactors and systems with immobilized biomass is awaited. The biocatalyst box (2) considers Who: biodiversity and its dynamic patterns, What: physiology, and Where: architecture creating substrate gradients. Particularly community dynamics and extracellular polymeric substances (EPS) still require insights. Performant OLAND (3) comprises fast start-up (storage possibility; fast growth of anammox bacteria), process stability (endured biomass retention; stress resilience), reasonable overall costs, high nitrogen removal efficiency and a low environmental footprint. Three important OLAND challenges are elaborated in detailed frameworks, demonstrating how to maximize nitrogen removal efficiency, minimize NO and N2O emissions and obtain through OLAND a plant-wide net energy gain from sewage treatment.

Source determination of nitrous oxide (N2O) from soils has so far been complicated by methodological constraints: the frequently used 15N tracer method could not differentiate between pathways related to nitrification, that is, nitrifier nitrification (NN), nitrifier denitrification (ND), and nitrification-coupled denitrification (NCD). To overcome this problem, a dual isotope method using both 15N and 18O was proposed. However, O exchange between nitrogen oxides and water has been found to disturb such a method. We here explain in detail a novel dual isotope method that allows to quantify O exchange in denitrification and to differentiate N2O production from NN, ND, NCD, and fertilizer denitrification (FD). The method has already been applied to a range of soils with good success. Potential of and scope for further improvement of the method are discussed

Source determination of nitrous oxide (N2O) from soils has so far been complicated by methodological constraints: the frequently used 15N tracer method could not differentiate between pathways related to nitrification, that is, nitrifier nitrification (NN), nitrifier denitrification (ND), and nitrification-coupled denitrification (NCD). To overcome this problem, a dual isotope method using both 15N and 18O was proposed. However, O exchange between nitrogen oxides and water has been found to disturb such a method. We here explain in detail a novel dual isotope method that allows to quantify O exchange in denitrification and to differentiate N2O production from NN, ND, NCD, and fertilizer denitrification (FD). The method has already been applied to a range of soils with good success. Potential of and scope for further improvement of the method are discussed

Microbial fuel cells (MFCs) have applications other than electricity production, including the capacity to power desirable reactions in the cathode chamber. However, current knowledge of the microbial ecology and physiology of biocathodes is minimal, and as a result more research dedicated to understanding the microbial communities active in cathode biofilms is required. Here we characterize the microbiology of denitrifying bacterial communities stimulated by reducing equivalents generated from the anodic oxidation of acetate. We analyzed biofilms isolated from two types of cathodic denitrification systems: (1) a loop format where the effluent from the carbon oxidation step in the anode is subjected to a nitrifying reactor which is fed to the cathode chamber and (2) an alternative non-loop format where anodic and cathodic feed streams are separated. The results of our study indicate the superior performance of the loop reactor in terms of enhanced current production and nitrate removal rates. We hypothesized that phylogenetic or structural features of the microbial communities could explain the increased performance of the loop reactor. We used PhyloChip with 16S rRNA (cDNA) and fluorescent in situ hybridization to characterize the active bacterial communities. Our study results reveal a greater richness, as well as an increased phylogenetic diversity, active in denitrifying biofilms than was previously identified in cathodic systems. Specifically, we identified Proteobacteria, Firmicutes and Chloroflexi members that were dominant in denitrifying cathodes. In addition, our study results indicate that it is the structural component, in terms of bacterial richness and evenness, rather than the phylogenetic affiliation of dominant bacteria, that best corresponds to cathode performance.

Nitrosomonas europaea is capable of incorporating exogenously supplied amino acids. Studies in whole cells revealed that at least eight amino acids are actively accumulated, probably by the action of three different transport systems, each with high affinity (mu-molar range) for several amino acids. Evidence for the action of secondary mechanisms of transport was obtained from efflux, counterflow and exchange experiments. More detailed information was obtained from studies in liposomes in which solubilized integral membrane proteins of N. europaea were incorporated. Uptake Of L-alanine in these liposomes could be driven by artificially imposed pH gradients and electrical potentials, but not by chemical sodium-ion gradients. These observations indicate that L-alanine is transported by a H+/alanine symport system. The ecological significance of secondary amino acid transport systems in autotrophic ammonium-oxidizing bacteria is discussed.

Autotrophic ammonia oxidizers depend on alkaline or neutral conditions for optimal activity. Below pH 7 growth and metabolic activity decrease dramatically. Actively oxidizing cells of Nitrosomonas europaea do not maintain a constant internal pH when the external pH is varied from 5 to 8. Studies of the kinetics and pH-dependency of ammonia and hydroxylamine oxidation by N. europaea revealed that hydroxylamine oxidation is moderately pH-sensitive, while ammonia oxidation decreases strongly with decreasing pH. Oxidation of these exogenous substrates results in the generation of higher proton motive force which is mainly composed of a DELTA-PSI. Hydroxylamine, but not ammonia, is oxidized at pH 5, which leads to the generation of a high proton motive force which drives energy-dependent processes such as ATP-synthesis and secondary transport of amino acids.Endogenous substrates can be oxidized between pH 5 to 8 and this results in the generation of a considerable proton motive force which is mainly composed of a DELTA-PSI. Inhibition of ammonia-mono-oxygenase or cytochrome aa3 does not influence the magnitude of this gradient or the oxygen consumption rate, indicating that endogenous respiration and ammonia oxidation are two distinct systems for energy transduction.The results indicate that the first step in ammonia oxidation is acid sensitive while the subsequent steps can take place and generate a proton motive force at acid pH.

Stable isotope analysis of oxygen (O) is increasingly used to determine the origin of nitrate (NO(3)-) and nitrous oxide (N(2)O) in the environment. The assumption underlying these studies is that the (18)O signature of NO(3)- and N(2)O provides information on the different O sources (O(2) and H(2)O) during the production of these compounds by various biochemical pathways. However, exchange of O atoms between H(2)O and intermediates of the (de)nitrification pathways may change the isotopic signal and thereby bias its interpretation for source determination. Chemical exchange of O between H(2)O and various nitrogenous oxides has been reported, but the probability and extent of its occurrence in terrestrial ecosystems remain unclear. Biochemical O exchange between H(2)O and nitrogenous oxides, NO(2)- in particular, has been reported for monocultures of many nitrifiers and denitrifiers that are abundant in nature, with exchange rates of up to 100%. Therefore, biochemical O exchange is likely to be important in most soil ecosystems, and should be taken into account in source determination studies. Failing to do so might lead to (i) an overestimation of nitrification as NO(3)- source, and (ii) an overestimation of nitrifier denitrification and nitrification-coupled denitrification as N(2)O production pathways. A method to quantify the rate and controls of biochemical O exchange in ecosystems is needed, and we argue this can only be done reliably with artificially enriched (18)O compounds. We conclude that in N source determination studies, the O isotopic signature of especially N(2)O should only be used with extreme caution.

Nitrifier denitrification is the reduction of NO2- to N2 by nitrifiers. It leads to the production of the greenhouse gas nitrous oxide (N2O) as an intermediate and possible end product. It is not known how important nitrifier denitrification is for the production of N2O in soils. We explored N2O production by nitrifier denitrification in relation to other N2O producing processes such as nitrification and denitrification under different soil conditions. The influence of aeration of the soil, different N sources, and pH were tested in four experiments. To differentiate between sources of N2O, an incubation method with inhibitors was used [Biol. Fertil. Soils 22 (1996) 331]. Sets of four incubations included controls without addition of inhibitors, incubations with addition of small concentrations of C2H2 (0.01–0.1 kPa), large concentrations of O2 (100 kPa), or a combination of C2H2 and O2. The results indicate that the availability of NO2- stimulated the apparent N2O production by nitrifier denitrification. A decreasing O2 content increased the total N2O production, but decreased N2O production by nitrifier denitrification. No significant effect of pH could be found. The study revealed problems concerning the use of the inhibitors C2H2 and O2. Almost one-third of all incubations with inhibitors produced more N2O than the controls. Possible reasons for the problems are discussed. The inhibitors C2H2 and O2 need to be tested thoroughly for their effects on different N2O producing processes before further application. [KEYWORDS: Nitrous oxide; Nitrifier denitrification; Nitrification; Denitrification; Acetylene; Oxygen; Inhibitors; Grasslands]

A procedure for start-up of oxygen-limited autotrophic nitrification-denitrification (OLAND) in a lab-scale rotating biological contactor (RBC) is presented. In this one-step process, NH4+ is directly converted to N2 without the need for an organic carbon source. The approach is based on a sequential addition of two types of easily available biocatalyst to the reactor during start-up: aerobic nitrifying and anaerobic, granular methanogenic sludge. The first is added as a source of aerobic ammonia-oxidizing bacteria (AAOB), the second as a possible source of planctomycetes including anaerobic ammonia-oxidizing bacteria (AnAOB). The initial nitrifying biofilm serves as a matrix for anaerobic cell incorporation. By subsequently imposing oxygen limitation, one can create an optimal environment for autotrophic N removal. In this way, N removal of about 250 mg of N L(-1) d(-1) was achieved after 100 d treating a synthetic NH4+-rich wastewater. By gradually imposing higher loads on the reactor, the N elimination could be increased to about 1.8 g of N L(-1) d(-1) at 250 d. The resulting microbial community was compared with that of the inocula using general bacterial and AAOB- and planctomycete-specific PCR primers. Subsequently, the RBC reactor was shown to treat a sludge digestor effluent under suboptimal and strongly varying conditions. The RBC biocatalyst was also submitted to complete absence of oxygen in a fixed-film bioreactor (FFBR) and proved able to remove NH4+ with NO2- as electron acceptor (maximal 434 mg of NH4+-N (g of VSS)(-1) d(-1) on day 136). DGGE and real-time PCR analysis demonstrated that the RBC biofilm was dominated by members of the genus Nitrosomonas and close relatives of Kuenenia stuttgartiensis, a known AnAOB. The latter was enriched during FFBR operation, but AAOB were still present and the ratio planctomycetes/AAOB rRNA gene copies was about 4.3 after 136 d of reactor operation. Whether this relates to an active role of AAOB in the anoxic N removal process remains to be solved.

We analysed the stable isotope composition of emitted N2O in a one-year field experiment (June 1998 to April 1999) in unfertilized controls, and after adding nitrogen by applying slurry or mineral N (calcium ammonium nitrate). Emitted N2O was analysed every 2¿4 weeks, with additional daily sampling for 10 days after each fertilizer application. In supplementary soil incubations, the isotopic composition of N2O was measured under defined conditions, favouring either denitrification or nitrification. Soil incubated for 48 h under conditions favouring nitrification emitted very little N2O (0.024 mol gdw ¿1) and still produced N2O from denitrification. Under denitrifying incubation conditions, much more N2O was formed (0.91 mol gdw ¿1 after 48 h). The isotope ratios of N2O emitted from denitrification stabilized at 15N = ¿40.8 ± 5.7 and 18O = 2.7 ± 6.3. In the field experiment, the N2O isotope data showed no clear seasonal trends or treatment effects. Annual means weighted by time and emission rate were 15N = ¿8.6 and 18O = 34.7 after slurry application, 15N = ¿4.6 and 18O = 24.0 after mineral fertilizer application and 15N = ¿6.4 and 18O = 35.6 in the control plots, respectively. So, in all treatments the emitted N2O was 15N-depleted compared to ambient air N2O (15N = 11.4 ± 11.6, 18O = 36.9 ± 10.7). Isotope analyses of the emitted N2O under field conditions per se allowed no unequivocal identification of the main N2O producing process. However, additional data on soil conditions and from laboratory experiments point to denitrification as the predominant N2O source. We concluded (1) that the isotope ratios of N2O emitted from the field soil were not only influenced by the source processes, but also by microbial reduction of N2O to N2 and (2) that N2O emission rates had to exceed 3.4 mol N2O m¿2 h¿1 to obtain reliable N2O isotope data

Nitrosomonas europaea is capable of incorporating exogenously supplied amino acids. Studies in whole cells revealed that at least eight amino acids are actively accumulated, probably by the action of three different transport systems, each with high affinity (mu-molar range) for several amino acids. Evidence for the action of secondary mechanisms of transport was obtained from efflux, counterflow and exchange experiments. More detailed information was obtained from studies in liposomes in which solubilized integral membrane proteins of N. europaea were incorporated. Uptake Of L-alanine in these liposomes could be driven by artificially imposed pH gradients and electrical potentials, but not by chemical sodium-ion gradients. These observations indicate that L-alanine is transported by a H+/alanine symport system. The ecological significance of secondary amino acid transport systems in autotrophic ammonium-oxidizing bacteria is discussed.

Nonylphenol (NP) is an estrogenic pollutant which is widely present in the aquatic environment. Biodegradation of NP can reduce the toxicological risk. In this study, aerobic biodegradation of NP in river sediment was investigated. The sediment used for the microcosm experiments was aged polluted with NP. The biodegradation of NP in the sediment occurred within 8 days with a lag phase of 2 days at 30 degrees C. During the biodegradation, nitro-nonylphenol metabolites were formed, which were further degraded to unknown compounds. The attached nitro-group originated from the ammonium in the medium. Five subsequent transfers were performed from original sediment and yielded a final stable population. In this NP-degrading culture, the microorganisms possibly involved in the biotransformation of NP to nitro-nonylphenol were related to ammonium-oxidizing bacteria. Besides the degradation of NP via nitro-nonylphenol, bacteria related to phenol-degrading species, which degrade phenol via ring cleavage, are abundantly present.

Autotrophic ammonia oxidizers depend on alkaline or neutral conditions for optimal activity. Below pH 7 growth and metabolic activity decrease dramatically. Actively oxidizing cells of Nitrosomonas europaea do not maintain a constant internal pH when the external pH is varied from 5 to 8. Studies of the kinetics and pH-dependency of ammonia and hydroxylamine oxidation by N. europaea revealed that hydroxylamine oxidation is moderately pH-sensitive, while ammonia oxidation decreases strongly with decreasing pH. Oxidation of these exogenous substrates results in the generation of higher proton motive force which is mainly composed of a DELTA-PSI. Hydroxylamine, but not ammonia, is oxidized at pH 5, which leads to the generation of a high proton motive force which drives energy-dependent processes such as ATP-synthesis and secondary transport of amino acids. Endogenous substrates can be oxidized between pH 5 to 8 and this results in the generation of a considerable proton motive force which is mainly composed of a DELTA-PSI. Inhibition of ammonia-mono-oxygenase or cytochrome aa3 does not influence the magnitude of this gradient or the oxygen consumption rate, indicating that endogenous respiration and ammonia oxidation are two distinct systems for energy transduction. The results indicate that the first step in ammonia oxidation is acid sensitive while the subsequent steps can take place and generate a proton motive force at acid pH.