Biofilm formation inhibition and dispersal of multi-species communities containing ammonia-oxidising bacteria.

Despite considerable research, the biofilm-forming capabilities of Nitrosomonas europaea are poorly understood for both mono and mixed-species communities. This study combined biofilm assays and molecular techniques to demonstrate that N. europaea makes very little biofilm on its own, and relies on the activity of associated heterotrophic bacteria to establish a biofilm. However, N. europaea has a vital role in the proliferation of mixed-species communities under carbon-limited conditions, such as in drinking water distribution systems, through the provision of organic carbon via ammonia oxidation. Results show that the addition of nitrification inhibitors to mixed-species nitrifying cultures under carbon-limited conditions disrupted biofilm formation and caused the dispersal of pre-formed biofilms. This dispersal effect was not observed when an organic carbon source, glucose, was included in the medium. Interestingly, inhibition of nitrification activity of these mixed-species biofilms in the presence of added glucose resulted in increased total biofilm formation compared to controls without the addition of nitrification inhibitors, or with only glucose added. This suggests that active AOB partially suppress or limit the overall growth of the heterotrophic bacteria. The experimental model developed here provides evidence that ammonia-oxidising bacteria (AOB) are involved in both the formation and maintenance of multi-species biofilm communities. The results demonstrate that the activity of the AOB not only support the growth and biofilm formation of heterotrophic bacteria by providing organic carbon, but also restrict and limit total biomass in mixed community systems.

Fate of Fecal Indicators in Resource-Oriented Sanitation Systems Using Nitrifying Bio-Treatment.

Hygienic fecal treatment in resource-oriented sanitation (ROS) systems is an important concern. Although the addition of nitrifying microorganisms is a sustainable fecal treatment method in ROS systems, it is essential to examine the cleanliness of this method. In this study, we investigated the fate of fecal indicators in source-separated fecal samples through tracking Escherichia coli and total coliforms. The effects of adding different amounts of Nitrosomonas europaea bio-seed, along with a constant amount of Nitrobacter winogradskyi bio-seed, were studied. In intact feces samples, the pathogen population underwent an initial increase, followed by a slight decrease, and eventually became constant. Although the addition of nitrifying microorganisms initially enhanced the pathogen growth rate, it caused the reduction process to become more efficient in the long-term. In addition to a constant concentration of 10,000 cells of N. winogradskyi per 1 g feces, a minimum amount of 3000 and 7000 cells of N. europaea per 1 g feces could completely remove E. coli and total coliforms, respectively, in less than 25 days. Increasing the amount of bio-seeds added can further reduce the time required for total pathogen removal.

Responses and recovery assessment of continuously cultured Nitrosomonas europaea under chronic ZnO nanoparticle stress: Effects of dissolved oxygen.

Although the antibacterial performances of emerging nanoparticles (NPs) have been extensively explored in the nitrifying systems, the impacts of dissolved oxygen (DO) levels on their bio-toxicities to the nitrifiers and the impaired cells' recovery potentials have seldom been addressed yet. In this study, the physiological and transcriptional responses of the typical ammonia oxidizers - Nitrosomonas europaea in a chemostat to the chronic ZnO NP exposure under different DO conditions were investigated. The results indicated that the cells in steady-growth state in the chemostat were more persevering than batch cultured ones to resist ZnO NP stress despite the dose-dependent NP inhibitory effects were observed. In addition, the occurred striking over-expressions of amoA and hao genes at the initial NP exposure stage suggested the cells' self-regulation potentials at the transcriptional level. The low DO (0.5 mg/L) cultured cells displayed higher sensitivity to NP stress than the high DO (2.0 mg/L) cultured ones, probably owning to the inefficient oxygen-dependent electron transfer from ammonia oxidation for energy conversion/production. The following 12-h NP-free batch recovery assays revealed that both high and low DO cultured cells possessed the physiological and metabolic activity recovery potentials, which were in negative correlation with the NP exposure time. The duration of NP stress and the resulting NP dissolution were critical for the cells' damage levels and their performance recoverability. The membrane preservation processes and the associated metabolism regulations were expected to actively participate in the cells' self-adaption to NP stress and thus be responsible for their metabolic activities recovery.

Nitrosomonas europaea biofilm formation is enhanced by Pseudomonas aeruginosa.

Biofilms are useful in biotechnology applications such as wastewater treatment, where aggregation of cells on surfaces can increase retention of slow-growing organisms such as ammonia-oxidizing bacteria (AOB). The formation and morphological development of polymicrobial biofilms including AOB are not thoroughly understood. Here, we investigated the formation of Nitrosomonas europaea AOB biofilms in flow cell systems. Nitrosomonas europaea developed substantially greater biovolume in co-culture with heterotrophic Pseudomonas aeruginosa than when cultured alone. In single-species biofilms, N. europaea formed thin, dispersed layers of cells. Contrastingly, when N. europaea was added to flow cells containing pre-established P. aeruginosa biofilms, N. europaea associated closely with P. aeruginosa, resulting in dual-species clusters with greater quantities of N. europaea. These results indicate that P. aeruginosa enhances the formation of N. europaea in biofilms. This favorable association of N. europaea with heterotrophic biofilms is expected to facilitate development of improved strategies for retention of N. europaea and other slow-growing AOB in engineered bioreactors.

Regulation of membrane fixation and energy production/conversion for adaptation and recovery of ZnO nanoparticle impacted Nitrosomonas europaea.

The ZnO nanoparticle (NP) effects on typical ammonia-oxidizing bacteria, Nitrosomonas europaea in a chemostat bioreactor, and the cells' toxicity adaptation and recovery potentials were explored. Hardly any inhibition was observed when the NP concentration was high up to 10 mg/L. The cells exposed to 50 mg/L ZnO NPs displayed time-dependent impairment and recovery potentials in terms of cell density, membrane integrity, nitrite production rate, and ammonia monooxygenase activity. The 6-h NP stress impaired cells were nearly completely restored during a 12-h recovery incubation, while the longer exposure time would cause irretrievable cell damage. Microarray analysis further indicated the transcriptional adaptation of N. europaea to NP stress. The regulations of genes encoding for membrane permeability or osmoprotectant, membrane integrity preservation, and inorganic ion transport during NP exposure and cell recovery revealed the importance of membrane fixation and the associated metabolisms for cells' self-protection and the following recovery from NP stress. The oxidative phosphorylation, carbon assimilation, and tricarboxylic acid (TCA) cycling pathways involved in the cells' antitoxicity activities and would promote the energy production/conversion efficiency for cell recovery. The heavy metal resistance, histidine metabolism, toxin-antitoxin defense, glycolysis, and sulfate reduction pathways were also suggested to participate in the cell detoxication and recovery processes. All these findings provided valuable insights into the mechanisms of cell-mediated ZnO NP cytotoxicity and their potential impacts on wastewater nitrogen removal system.

Growth modelling of Nitrosomonas europaea ATCC® 19718 and Nitrobacter winogradskyi ATCC® 25391: A new online indicator of the partial nitrification.

The aim of the present work was to study the growth of two nitrifying bacteria. For modelling the nitrifying subsystem of the MELiSSA loop, Nitrosomonas europaea ATCC® 19718 and Nitrobacter winogradskyi ATCC® 25931 were grown separately and in cocultures. The kinetic parameters of a stoichiometric mass balanced Pirt model were identified: µmax=0.054h(-1), decay rate b=0.003h(-1) and maintenance rate m=0.135gN-NH4(+)·gX(-1)·h(-1) for Nitrosomonas europaea; µmax=0.024h(-1), b=0.001h(-1) and m=0.467gN-NO2(-)·gX(-1)·h(-1) for Nitrobacter winogradskyi. A predictive structured model of nitrification in co-culture was developed. The online evolution of the addition of KOH is correlated to the nitritation; the dissolved oxygen concentration is correlated to both nitritation and nitratation. The model suitably represents these two variables so that transient partial nitrification is assessed. This is a clue for avoiding partial nitrification by predictive functional control.

Optimization of fertilization characteristics of urine by addition of Nitrosomonas europaea bio-seed.

BACKGROUND: Because of the high concentration of nutrients in human urine, its utilization as an organic fertilizer has been notable throughout history. However, the nitrogen compounds in urine are not stable. Therefore, to convert urine into a suitable fertilizer, it is important to stabilize and adjust unstable nitrogen compounds such as ammonia. Because nitrification can influence the nitrogen profile, the use of nitrifying microorganisms can be useful for stabilizing the nitrogen profile of urine. This study investigated the changes in nitrogen compounds in pure urine and examined the effect of adding Nitrosomonas europaea bio-seed solution on these changes. RESULTS: It was found that the addition of bio-seed could reduce nitrogen loss as well as the time required to stabilize the nitrogen profile. Furthermore, the optimum concentration of bio-seed (6 × 10(5) N. europaea cells L(-1) ) that not only leads to the least nutrient loss but also results in an adequate nitrate/ammonium ratio and regulates the amount of nitrate produced, thereby preventing over-fertilization, was determined. CONCLUSION: At this concentration, no dilution or dewatering is required, thus minimizing water and energy consumption. Usage of the optimum of concentration of bio-seed will also eliminate the need for inorganic chemical additives. © 2016 Society of Chemical Industry.

Steady-State Growth under Inorganic Carbon Limitation Conditions Increases Energy Consumption for Maintenance and Enhances Nitrous Oxide Production in Nitrosomonas europaea.

UNLABELLED: Nitrosomonas europaea is a chemolithoautotrophic bacterium that oxidizes ammonia (NH3) to obtain energy for growth on carbon dioxide (CO2) and can also produce nitrous oxide (N2O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses of N. europaea to conditions of replete (>5.2 mM) and limited inorganic carbon (IC) provided by either 1.0 mM or 0.2 mM sodium carbonate (Na2CO3) supplemented with atmospheric CO2 IC-limited cultures oxidized 25 to 58% of available NH3 to nitrite, depending on the dilution rate and Na2CO3 concentration. IC limitation resulted in a 2.3-fold increase in cellular maintenance energy requirements compared to those for NH3-limited cultures. Rates of N2O production increased 2.5- and 6.3-fold under the two IC-limited conditions, increasing the percentage of oxidized NH3-N that was transformed to N2O-N from 0.5% (replete) up to 4.4% (0.2 mM Na2CO3). Transcriptome analysis showed differential expression (P ≤ 0.05) of 488 genes (20% of inventory) between replete and IC-limited conditions, but few differences were detected between the two IC-limiting treatments. IC-limited conditions resulted in a decreased expression of ammonium/ammonia transporter and ammonia monooxygenase subunits and increased the expression of genes involved in C1 metabolism, including the genes for RuBisCO (cbb gene cluster), carbonic anhydrase, folate-linked metabolism of C1 moieties, and putative C salvage due to oxygenase activity of RuBisCO. Increased expression of nitrite reductase (gene cluster NE0924 to NE0927) correlated with increased production of N2O. Together, these data suggest that N. europaea adapts physiologically during IC-limited steady-state growth, which leads to the uncoupling of NH3 oxidation from growth and increased N2O production. IMPORTANCE: Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, is an important process in the global nitrogen cycle. This process is generally dependent on ammonia-oxidizing microorganisms and nitrite-oxidizing bacteria. Most nitrifiers are chemolithoautotrophs that fix inorganic carbon (CO2) for growth. Here, we investigate how inorganic carbon limitation modifies the physiology and transcriptome of Nitrosomonas europaea, a model ammonia-oxidizing bacterium, and report on increased production of N2O, a potent greenhouse gas. This study, along with previous work, suggests that inorganic carbon limitation may be an important factor in controlling N2O emissions from nitrification in soils and wastewater treatment.

High cell density cultivation of the chemolithoautotrophic bacterium Nitrosomonas europaea.

Nitrosomonas europaea is a chemolithoautotrophic nitrifier, a gram-negative bacterium that can obtain all energy required for growth from the oxidation of ammonia to nitrite, and this may be beneficial for various biotechnological and environmental applications. However, compared to other bacteria, growth of ammonia oxidizing bacteria is very slow. A prerequisite to produce high cell density N. europaea cultures is to minimize the concentrations of inhibitory metabolic by-products. During growth on ammonia nitrite accumulates, as a consequence, N. europaea cannot grow to high cell concentrations under conventional batch conditions. Here, we show that single-vessel dialysis membrane bioreactors can be used to obtain substantially increased N. europaea biomasses and substantially reduced nitrite levels in media initially containing high amounts of the substrate. Dialysis membrane bioreactor fermentations were run in batch as well as in continuous mode. Growth was monitored with cell concentration determinations, by assessing dry cell mass and by monitoring ammonium consumption as well as nitrite formation. In addition, metabolic activity was probed with in vivo acridine orange staining. Under continuous substrate feed, the maximal cell concentration (2.79 × 10(12)/L) and maximal dry cell mass (0.895 g/L) achieved more than doubled the highest values reported for N. europaea cultivations to date.

Planktonic and biofilm-grown nitrogen-cycling bacteria exhibit different susceptibilities to copper nanoparticles.

Proper characterization of nanoparticle (NP) interactions with environmentally relevant bacteria under representative conditions is necessary to enable their sustainable manufacture, use, and disposal. Previous nanotoxicology research based on planktonic growth has not adequately explored biofilms, which serve as the predominant mode of bacterial growth in natural and engineered environments. Copper nanoparticle (Cu-NP) impacts on biofilms were compared with respective planktonic cultures of the ammonium-oxidizing Nitrosomonas europaea, nitrogen-fixing Azotobacter vinelandii, and denitrifying Paracoccus denitrificans using a suite of independent toxicity diagnostics. Median inhibitory concentration (IC50) values derived from adenosine triphosphate (ATP) for Cu-NPs were lower in N. europaea biofilms (19.6 ± 15.3 mg/L) than in planktonic cells (49.0 ± 8.0 mg/L). However, in absorbance-based growth assays, compared with unexposed controls, N. europaea growth rates in biofilms were twice as resilient to inhibition than those in planktonic cultures. Similarly, relative to unexposed controls, growth rates and yields of P. denitrificans in biofilms exposed to Cu-NPs were 40-fold to 50-fold less inhibited than those in planktonic cells. Physiological evaluation of ammonium oxidation and nitrate reduction suggested that biofilms were also less inhibited by Cu-NPs than planktonic cells. Furthermore, functional gene expression for ammonium oxidation (amoA) and nitrite reduction (nirK) showed lower inhibition by NPs in biofilms relative to planktonic-grown cells. These results suggest that biofilms mitigate NP impacts, and that nitrogen-cycling bacteria in wastewater, wetlands, and soils might be more resilient to NPs than planktonic-based assessments suggest.

Interactions of Nitrosomonas europaea and Nitrobacter winogradskyi grown in co-culture.

Nitrosomonas europaea and Nitrobacter winogradskyi were grown singly and in co-culture in chemostats to probe for physiological differences between the two growth conditions. Co-culture growth medium containing 60 mM NH4 (+) resulted in a cell density (0.20-0.29 OD600) greater than the sum of the densities in single chemostat cultures, i.e., 0.09-0.14 OD600 for N. europaea with 60 mM NH4 (+)and 0.04-0.06 OD600 for N. winogradskyi with 60 mM NO2 (-). The NO2 (-)- and NH4 (+)-dependent O2 uptake rates, qRT-PCR, and microscopic observations indicated that in co-culture, N. europaea contributed ~0.20 OD600 (~80 %) and N. winogradskyi ~0.05 OD600 (~20 %). In co-culture, the transcriptomes showed that the mRNA levels of 773 genes in N. europaea (30.2 % of the genes) and of 372 genes in N. winogradskyi (11.8 % of the genes) changed significantly. Total cell growth and the analysis of the transcriptome revealed that in co-culture, N. europaea benefits more than N. winogradskyi.

Kinetic parameter estimation in N. europaea biofilms using a 2-D reactive transport model.

Biofilms of the ammonia oxidizing bacterium Nitrosomonas europaea were cultivated to study microbial processes associated with ammonia oxidation in pure culture. We explored the hypothesis that the kinetic parameters of ammonia oxidation in N. europaea biofilms were in the range of those determined with batch suspended cells. Oxygen and pH microelectrodes were used to measure dissolved oxygen (DO) concentrations and pH above and inside biofilms and reactive transport modeling was performed to simulate the measured DO and pH profiles. A two dimensional (2-D) model was used to simulate advection parallel to the biofilm surface and diffusion through the overlying fluid while reaction and diffusion were simulated in the biofilm. Three experimental studies of microsensor measurements were performed with biofilms: i) NH3 concentrations near the Ksn value of 40 µM determined in suspended cell tests ii) Limited buffering capacity which resulted in a pH gradient within the biofilms and iii) NH3 concentrations well below the Ksn value. Very good fits to the DO concentration profiles both in the fluid above and in the biofilms were achieved using the 2-D model. The modeling study revealed that the half-saturation coefficient for NH3 in N. europaea biofilms was close to the value measured in suspended cells. However, the third study of biofilms with low availability of NH3 deviated from the model prediction. The model also predicted shifts in the DO profiles and the gradient in pH that resulted for the case of limited buffering capacity. The results illustrate the importance of incorporating both key transport and chemical processes in a biofilm reactive transport model.

Hydroxylamine addition impact to Nitrosomonas europaea activity in the presence of monochloramine.

In drinking water, monochloramine may promote ammonia­oxidizing bacteria (AOB) growth because of concurrent ammonia presence. AOB use (i) ammonia monooxygenase for biological ammonia oxidation to hydroxylamine and (ii) hydroxylamine oxidoreductase for biological hydroxylamine oxidation to nitrite. In addition, monochloramine and hydroxylamine abiotically react, providing AOB a potential benefit by removing the disinfectant (monochloramine) and releasing growth substrate (ammonia). Alternatively and because biological hydroxylamine oxidation supplies the electrons (reductant) required for biological ammonia oxidation, the monochloramine/hydroxylamine abiotic reaction represents a possible inactivation mechanism by consuming hydroxylamine and inhibiting reductant generation. To investigate the abiotic monochloramine and hydroxylamine reaction's impact on AOB activity, the current study used batch experiments with Nitrosomonas europaea (AOB pure culture), ammonia, monochloramine, and hydroxylamine addition. To decipher whether hydroxylamine addition benefitted N. europaea activity by (i) removing monochloramine and releasing free ammonia or (ii) providing an additional effect (possibly the aforementioned reductant source), a previously developed cometabolism model was coupled with an abiotic monochloramine and hydroxylamine model for data interpretation. N. europaea maintained ammonia oxidizing activity when hydroxylamine was added before complete ammonia oxidation cessation. The impact could not be accounted for by monochloramine removal and free ammonia release alone and was concentration dependent for both monochloramine and hydroxylamine. In addition, a preferential negative impact occurred for ammonia versus hydroxylamine oxidation. These results suggest an additional benefit of exogenous hydroxylamine addition beyond monochloramine removal and free ammonia release, possibly providing reductant generation.

Application of an integrated statistical design for optimization of culture condition for ammonium removal by Nitrosomonas europaea.

Statistical methodology was applied to the optimization of the ammonium oxidation by Nitrosomonas europaea for biomass concentration (C(B)), nitrite yield (Y(N)) and ammonium removal (R(A)). Initial screening by Plackett-Burman design was performed to select major variables out of nineteen factors, among which NH4Cl concentration (C(N)), trace element solution (TES), agitation speed (AS), and fermentation time (T) were found to have significant effects. Path of steepest ascent and response surface methodology was applied to optimize the levels of the selected factors. Finally, multi-objective optimization was used to obtain optimal condition by compromise of the three desirable objectives through a combination of weighted coefficient method coupled with entropy measurement methodology. These models enabled us to identify the optimum operation conditions (C(N)= 84.1 mM; TES = 0.74 ml; AS= 100 rpm and T = 78 h), under which C(B)= 3.386×10(8) cells/ml; Y(N)= 1.98 mg/mg and R(A) = 97.76% were simultaneously obtained. The optimized conditions were shown to be feasible through verification tests.

High-rate, high-yield production of methanol by ammonia-oxidizing bacteria.

The overall goal of this study was to develop an appropriate biological process for achieving autotrophic conversion of methane (CH(4)) to methanol (CH3OH). In this study, we employed ammonia-oxidizing bacteria (AOB) to selectively and partially oxidize CH(4) to CH(3)OH. In fed-batch reactors using mixed nitrifying enrichment cultures from a continuous bioreactor, up to 59.89 ± 1.12 mg COD/L of CH(3)OH was produced within an incubation time of 7 h, which is approximately ten times the yield obtained previously using pure cultures of Nitrosomonas europaea. The maximum specific rate of CH(4) to CH(3)OH conversion obtained during this study was 0.82 mg CH(3)OH COD/mg AOB biomass COD-d, which is 1.5 times the highest value reported with pure cultures. Notwithstanding these positive results, CH(4) oxidation to CH(3)OH by AOB was inhibited by NH(3) (the primary substrate for the oxidative enzyme, ammonia monooxygenase, AMO) as well as the product, CH(3)OH, itself. Further, oxidation of CH(4) to CH(3)OH by AOB was also limited by reducing equivalents supply, which could be overcome by externally supplying hydroxylamine (NH(2)OH) as an electron donor. Therefore, a potential optimum design for promoting CH(4) to CH(3)OH oxidation by AOB could involve supplying NH(3) (needed to maintain AMO activity) uncoupled from the supply of NH(2)OH and CH(4). Partial oxidation of CH(4)-containing gases to CH3OH by AOB represents an attractive platform for the conversion of a gaseous mixture to an aqueous compound, which could be used as a commodity chemical. Alternately, the nitrate and CH(3) OH thus produced could be channeled to a downstream anoxic zone in a biological nitrogen removal process to effect nitrate reduction to N(2), using an internally produced organic electron donor.

Axenic cultures of Nitrosomonas europaea and Nitrobacter winogradskyi in autotrophic conditions: a new protocol for kinetic studies.

As a part of a natural biological N-cycle, nitrification is one of the steps included in the conception of artificial ecosystems designed for extraterrestrial life support systems (LSS) such as Micro-Ecological Life Support System Alternative (MELiSSA) project, which is the LSS project of the European Space Agency. Nitrification in aerobic environments is carried out by two groups of bacteria in a two-step process. The ammonia-oxidizing bacteria (Nitrosomonas europaea) realize the oxidation of ammonia to nitrite, and the nitrite-oxidizing bacteria (Nitrobacter winogradskyi), the oxidation of nitrite to nitrate. In both cases, the bacteria achieve these oxidations to obtain an energy and reductant source for their growth and maintenance. Furthermore, both groups also use CO2 predominantly as their carbon source. They are typically found together in ecosystems, and consequently, nitrite accumulation is rare. Due to the necessity of modeling accurately conversion yields and transformation rates to achieve a complete modeling of MELiSSA, the present study focuses on the experimental determination of nitrogen to biomass conversion yields. Kinetic and mass balance studies for axenic cultures of Nitrosomonas europaea and Nitrobacter winogradskyi in autotrophic conditions are performed. The follow-up of these cultures is done using flow cytometry for assessing biomass concentrations and ionic chromatography for ammonium, nitrite, and nitrate concentrations. A linear correlation is observed between cell count and optical density (OD) measurement (within a 10 % accuracy) validating OD measurements for an on-line estimation of biomass quantity even at very low biomass concentrations. The conversion between cell count and biomass concentration has been determined: 7.1 × 10¹² cells g dry matter (DM)⁻¹ for Nitrobacter and 6.3 × 10¹² cells g DM⁻¹ for Nitrosomonas. Nitrogen substrates and products are assessed redundantly showing excellent agreement for mass balance purposes and conversion yields determination. Although the dominant phenomena are the oxidation of NH4⁺ into nitrite (0.95 mol mol N⁻¹ for Nitrosomonas europaea within an accuracy of 3 %) and nitrite into nitrate (0.975 mol mol N⁻¹ for Nitrobacter winogradskyi within an accuracy of 2 %), the Nitrosomonas europaea conversion yield is estimated to be 0.42 g DM mol N⁻¹, and Nitrobacter winogradskyi conversion yield is estimated to be 0.27 g DM mol N⁻¹. The growth rates of both strains appear to be dominated by the oxygen transfer into the experimental setups.

Global analysis of the Nitrosomonas europaea iron starvation stimulon.

The importance of iron to the metabolism of the ammonia-oxidizing bacterium Nitrosomonas europaea is well known. However, the mechanisms by which N. europaea acquires iron under iron limitation are less well known. To obtain insight into these mechanisms, transcriptional profiling of N. europaea was performed during growth under different iron availabilities. Of 2,355 N. europaea genes on DNA microarrays, transcripts for 247 genes were identified as differentially expressed when cells were grown under iron limitation compared to cells grown under iron-replete conditions. Genes with higher transcript levels in response to iron limitation included those with confirmed or assigned roles in iron acquisition. Genes with lower transcript levels included those encoding iron-containing proteins. Our analysis identified several potentially novel iron acquisition systems in N. europaea and provided support for the primary involvement of a TonB-dependent heme receptor gene in N. europaea iron homeostasis. We demonstrated that hemoglobin can act as an iron source under iron-depleted conditions for N. europaea. In addition, we identified a hypothetical protein carrying a lipocalin-like domain that may have the ability to chelate iron for growth in iron-limited media.

Investigating Nitrosomonas europaea stress biomarkers in batch, continuous culture, and biofilm reactors.

The understanding of nitrification inhibition in ammonia oxidizing bacteria (AOB) by priority pollutants and emerging contaminants is critical in managing the nitrogen cycle to preserve current water supplies, one of the National Academy of Engineers Grand Challenges in Engineering for the twenty-first century. Nitrosomonas europaea is an excellent model AOB for nitrification inhibition experimentation due to its well-defined NH(3) metabolism and the availability of a wide range of physiological and transcriptional tools that can characterize the mechanism of nitrification inhibition and probe N. europaea's response to the inhibitor. This chapter is a compilation of the physiological and transcriptional methods that have been used to characterize nitrification inhibition of N. europaea under a wide variety of growth conditions including batch, continuously cultured, and in biofilms. The protocols presented here can be applied to other AOB, and may be readily adapted for other autotrophic bacteria (e.g., nitrite oxidizing bacteria).

Nitrogen metabolism and kinetics of ammonia-oxidizing archaea.

The discovery of ammonia-oxidizing mesophilic and thermophilic Group I archaea changed the century-old paradigm that aerobic ammonia oxidation is solely mediated by two small clades of Beta- and Gammaproteobacteria. Group I archaea are extremely diverse and ubiquitous in marine and terrestrial environments, accounting for 20-30% of the microbial plankton in the global oceans. Recent studies indicated that many of these organisms carry putative ammonia monooxygenase genes and are more abundant than ammonia-oxidizing bacteria in most natural environments suggesting a potentially significant role in the nitrogen cycle. The isolation of Nitrosopumilus maritimus strain SCM1 provided the first direct evidence that Group I archaea indeed gain energy from ammonia oxidation. To characterize the physiology of this archaeal nitrifier, we developed a respirometry setup particularly suited for activity measurements in dilute microbial cultures with extremely low oxygen uptake rates. Here, we describe the setup and review the kinetic experiments conducted with N. maritimus and other nitrifying microorganisms. These experiments demonstrated that N. maritimus is adapted to grow on ammonia concentrations found in oligotrophic open ocean environments, far below the survival threshold of ammonia-oxidizing bacteria. The described setup and experimental procedures should facilitate physiological studies on other nitrifying archaea and oligotrophic microorganisms in general.