Competition between Ammonia-Oxidizing Archaea and Bacteria from Freshwater Environments.

In the environment, nutrients are rarely available in a constant supply. Therefore, microorganisms require strategies to compete for limiting nutrients. In freshwater systems, ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) compete with heterotrophic bacteria, photosynthetic microorganisms, and each other for ammonium, which AOA and AOB utilize as their sole source of energy and nitrogen. We investigated the competition between highly enriched cultures of AOA (AOA-AC1) and AOB (AOB-G5-7) for ammonium. Based on the amoA gene, the newly enriched archaeal ammonia oxidizer in AOA-AC1 was closely related to Nitrosotenuis spp., and the bacterial ammonia oxidizer in AOB-G5-7, Nitrosomonas sp. strain Is79, belonged to the Nitrosomonas oligotropha group (Nitrosomonas cluster 6a). Growth experiments in batch cultures showed that AOB-G5-7 had higher growth rates than AOA-AC1 at higher ammonium concentrations. During chemostat competition experiments under ammonium-limiting conditions, AOA-AC1 dominated the cultures, while AOB-G5-7 decreased in abundance. In batch cultures, the outcome of the competition between AOA and AOB was determined by the initial ammonium concentrations. AOA-AC1 was the dominant ammonia oxidizer at an initial ammonium concentration of 50 µM, and AOB-G5-7 was dominant at 500 µM. These findings indicate that during direct competition, AOA-AC1 was able to use ammonium that was unavailable to AOB-G5-7, while AOB-G5-7 dominated at higher ammonium concentrations. The results are in strong accordance with environmental survey data suggesting that AOA are mainly responsible for ammonia oxidation under more oligotrophic conditions, whereas AOB dominate under eutrophic conditions. IMPORTANCE Nitrification is an important process in the global nitrogen cycle. The first step, ammonia oxidation to nitrite, can be carried out by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). In many natural environments, these ammonia oxidizers coexist. Therefore, it is important to understand the population dynamics in response to increasing ammonium concentrations. Here, we study the competition between AOA and AOB enriched from freshwater systems. The results demonstrate that AOA are more abundant in systems with low ammonium availabilities and that AOB are more abundant when the ammonium availability increases. These results will help to predict potential shifts in the community composition of ammonia oxidizers in the environment due to changes in ammonium availability.

Transcriptome Analysis of the Ammonia-Oxidizing Bacterium Nitrosomonas mobilis Ms1 Reveals Division of Labor between Aggregates and Free-living Cells.

Bacteria change their metabolic states to increase survival by forming aggregates. Ammonia-oxidizing bacteria also form aggregates in response to environmental stresses. Nitrosomonas mobilis, an ammonia-oxidizing bacterium with high stress tolerance, often forms aggregates mainly in wastewater treatment systems. Despite the high frequency of aggregate formation by N. mobilis, its relationship with survival currently remains unclear. In the present study, aggregates were formed in the late stage of culture with the accumulation of nitrite as a growth inhibitor. To clarify the significance of aggregate formation in N. mobilis Ms1, a transcriptome analysis was performed. Comparisons of the early and late stages of culture revealed that the expression of stress response genes (chaperones and proteases) increased in the early stage. Aggregate formation may lead to stress avoidance because stress response genes were not up-regulated in the late stage of culture during which aggregates formed. Furthermore, comparisons of free-living cells with aggregates in the early stage of culture showed differences in gene expression related to biosynthesis (ATP synthase and ribosomal proteins) and motility and adhesion (flagella, pilus, and chemotaxis). Biosynthesis genes for growth were up-regulated in free-living cells, while motility and adhesion genes for adaptation were up-regulated in aggregates. These results indicate that N. mobilis Ms1 cells adapt to an unfavorable environment and grow through the division of labor between aggregates and free-living cells.

The drought and high wet soil condition impact on PAH (phenanthrene) toxicity towards nitrifying bacteria.

A few previous studies showed that the low soil moisture could interact with the toxic effect of the polycyclic aromatic hydrocarbons (PAHs) towards animals (mostly invertebrates). In the present research the impact of the soil moisture in the wide range (from the drought to high moisture conditions) in three different soil materials on toxic effect of the PAH (phenanthrene) towards soil microorganisms (nitrifying bacteria activity) was evaluated. The three dry soil materials were artificially contaminated with phenanthrene (0, 1, 10, 100 and 1000 mg kg-1 dry mass of soil) and moistened to the varied levels of the soil moisture (30% WHC (dry), 55% WHC (optimal) and 80% WHC (highly wet conditions)). After 7 days incubation, the nitrification potential was measured. The results of the proposed ANCOVA multiple regression model (adjusted R2 = 0.91), showed that the increase of soil moisture enhanced the toxicity of the phenanthrene towards nitrification potential and this combined moisture-phenanthrene effect was soil dependent. Therefore, the effect of the soil moisture in combination with the soil diversity should not be missed in the ecotoxicological risk assessment of the PAHs.

Impact of Heavy Metal Pollution on Ammonia Oxidizers in Soils in the Vicinity of a Tailings Dam, Baotou, China.

Soil heavy metal pollution has received increasing attention due to their toxicity to soil microorganisms. We have analyzed the effects of heavy metal pollution on ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in soils in the vicinity of a tailings dam of Baotou region, China. Results showed that AOB were dominated with Nitrosomonas-like clusters, while AOA was dominated by group1.1b (Nitrososphaera cluster). Single Cd and Cr contents, as well as compound heavy metal pollution levels, had a significant negative impact on soil potential nitrification rate and both diversities of AOA and AOB. No clear relationship was found between any single heavy metal and abundance of AOA or AOB. But compound pollution could significantly decrease AOA abundance. The results indicated that heavy metal pollution had an obviously deleterious effect on the abundance, diversity, activity and composition of ammonia oxidizers in natural soils.

Mixed nitrifying bacteria culture under different temperature dropping strategies: Nitrification performance, activity, and community.

In this study, the nitrification performance, metabolic activity, antioxidant enzyme activity as well as bacterial community of mixed nitrifying bacteria culture under different temperature dropping strategies [(#1) growth temperature kept at 20 °C; (#2) sharp1 decreased from 20 °C to 10 °C; (#3) growth at 20 °C for 6 days followed by sharp decrease to 10 °C; and (#4) gradual decreased from 20 °C to 10 °C] were evaluated. It was shown that acclimation at 20 °C for 6 days allowed to maintain better nitrification activity at 10 °C. The nitrite oxidation capacity of nitrifiers was significantly correlated with the relative light unit (RLU) (p < .05) and the fluctuation of superoxide dismutase (SOD) enzyme activity (p < .01). With serial #3 showed the highest RLU levels and the least SOD enzyme fluctuation as compared to serials #2 and #4. Throughout the experimental period, Nitrosospira and Nitrosomonas as well as Nitrospira were identified as the predominant ammonia-oxidizing bacteria (AOB) and nitrate-oxidizing bacteria (NOB). The dynamic change of AOB/NOB ratios and nitrification activity in serials #2-#4 demonstrated that AOB recovered better than NOB with long-term 10 °C exposure, and the nitrification performance was mainly limited by the nitrite oxidation capacity of NOB. Applying 6 days acclimation at 20 °C was beneficial for the mixed nitrifying bacteria culture to cope with low temperature (10 °C) stress, possibly due to the maintenance of metabolic activity, antioxidant enzyme activity stability as well as appropriate AOB/NOB ratio.

Modeling Nitrogen Dynamics in a Waste Stabilization Pond System Using Flexible Modeling Environment with MCMC.

This study presents an approach for obtaining realization sets of parameters for nitrogen removal in a pilot-scale waste stabilization pond (WSP) system. The proposed approach was designed for optimal parameterization, local sensitivity analysis, and global uncertainty analysis of a dynamic simulation model for the WSP by using the R software package Flexible Modeling Environment (R-FME) with the Markov chain Monte Carlo (MCMC) method. Additionally, generalized likelihood uncertainty estimation (GLUE) was integrated into the FME to evaluate the major parameters that affect the simulation outputs in the study WSP. Comprehensive modeling analysis was used to simulate and assess nine parameters and concentrations of ON-N, NH3-N and NO3-N. Results indicate that the integrated FME-GLUE-based model, with good Nash-Sutcliffe coefficients (0.53-0.69) and correlation coefficients (0.76-0.83), successfully simulates the concentrations of ON-N, NH3-N and NO3-N. Moreover, the Arrhenius constant was the only parameter sensitive to model performances of ON-N and NH3-N simulations. However, Nitrosomonas growth rate, the denitrification constant, and the maximum growth rate at 20 °C were sensitive to ON-N and NO3-N simulation, which was measured using global sensitivity.

Distribution and genetic diversity of microbial populations in the pilot-scale biofilter for simultaneous removal of ammonia, iron and manganese from real groundwater.

A pilot-scale biofilter treating real groundwater was developed in this study, which showed that ammonia, iron and manganese were mainly removed at 0.4, 0.4 and 0.8 m of the filter bed, respectively, and the corresponding removal efficiencies were 90.82%, 95.48% and 95.90% in steady phase, respectively. The variation of microbial populations in the biofilter during start-up process was also investigated using high-throughput pyrosequencing (HTP). Results indicated that the main functional microbes for ammonia, iron and manganese removal were Nitrosomonas, Crenothrix and Crenothrix, respectively, which was mainly distributed at 0.8, 0, and 0.8 m of the filter bed with a corresponding abundance of 8.7%, 28.12% and 11.33% in steady phase, respectively. Kinds of other bacteria which may be related to methane, hydrogen sulfide and organic matter removal, were also found. In addition, small part of archaea was also detected, such as Candidatus Nitrososphaera, which plays a role in nitritation.

Start-up and bacterial community compositions of partial nitrification in moving bed biofilm reactor.

Partial nitrification (PN) has been considered as one of the promising processes for pretreatment of ammonium-rich wastewater. In this study, a kind of novel carriers with enhanced hydrophilicity and electrophilicity was implemented in a moving bed biofilm reactor (MBBR) to start up PN process. Results indicated that biofilm formation rate was higher on modified carriers. In comparison with the reactor filled with traditional carriers (start-up period of 21 days), it took only 14 days to start up PN successfully with ammonia removal efficiency and nitrite accumulation rate of 90 and 91%, respectively, in the reactor filled with modified carriers. Evident changes of spatial distributions and community structures had been detected during the start-up. Free-floating cells existed in planktonic sludge, while these microorganisms trended to form flocs in the biofilm. High-throughput pyrosequencing results indicated that Nitrosomonas was the predominant ammonia-oxidizing bacterium (AOB) in the PN system, while Comamonas might also play a vital role for nitrogen oxidation. Additionally, some other bacteria such as Ferruginibacter, Ottowia, Saprospiraceae, and Rhizobacter were selected to establish stable footholds. This study would be potentially significant for better understanding the microbial features and developing efficient strategies accordingly for MBBR-based PN operation.

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.

Partial nitrification and denitrification of mature landfill leachate using a pilot-scale continuous activated sludge process at low dissolved oxygen.

Controlling of low dissolved oxygen (DO) levels (0.1-0.5mg/L), a cost-effective strategy, was applied to a pilot-scale anoxic-oxic-oxic-anoxic process for partial nitrification and denitrification of mature landfill leachate. High ammonium removal efficiency, stable nitrite accumulation rate and total nitrogen removal efficiency was higher than 95.0%, 90.0% and 66.4%, respectively, implying potential application of this process for nitrogen removal of mature landfill leachate. Efficient nitrite accumulation in the first oxic reactor depended on low DO conditions and sufficient alkalinity. However, operational limit was mainly decided by actual hydraulic retention time (AHRT) of the first oxic reactor and appeared with AHRT less than 13.9h under DO of 0.3-0.5mg/L. High-throughput sequencing analysis demonstrated significant change of bacterial diversity in the first oxic reactor after a long-term operation and dominant bacteria genus Nitrosomonas was shown to be responsible for NH4(+)-N removal and nitrite accumulation under low DO levels.

Effects of Bacterial Community Members on the Proteome of the Ammonia-Oxidizing Bacterium Nitrosomonas sp. Strain Is79.

UNLABELLED: Microorganisms in the environment do not exist as the often-studied pure cultures but as members of complex microbial communities. Characterizing the interactions within microbial communities is essential to understand their function in both natural and engineered environments. In this study, we investigated how the presence of a nitrite-oxidizing bacterium (NOB) and heterotrophic bacteria affect the growth and proteome of the chemolithoautotrophic ammonia-oxidizing bacterium (AOB) Nitrosomonas sp. strain Is79. We investigated Nitrosomonas sp. Is79 in co-culture with Nitrobacter winogradskyi, in co-cultures with selected heterotrophic bacteria, and as a member of the nitrifying enrichment culture G5-7. In batch culture, N. winogradskyi and heterotrophic bacteria had positive effects on the growth of Nitrosomonas sp. Is79. An isobaric tag for relative and absolute quantification (iTRAQ) liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach was used to investigate the effect of N. winogradskyi and the co-cultured heterotrophic bacteria from G5-7 on the proteome of Nitrosomonas sp. Is79. In co-culture with N. winogradskyi, several Nitrosomonas sp. Is79 oxidative stress response proteins changed in abundance, with periplasmic proteins increasing and cytoplasmic proteins decreasing in abundance. In the presence of heterotrophic bacteria, the abundance of proteins directly related to the ammonia oxidation pathway increased, while the abundance of proteins related to amino acid synthesis and metabolism decreased. In summary, the proteome of Nitrosomonas sp. Is79 was differentially influenced by the presence of either N. winogradskyi or heterotrophic bacteria. Together, N. winogradskyi and heterotrophic bacteria reduced the oxidative stress for Nitrosomonas sp. Is79, which resulted in more efficient metabolism. IMPORTANCE: Aerobic ammonia-oxidizing microorganisms play an important role in the global nitrogen cycle, converting ammonia to nitrite. In their natural environment, they coexist and interact with nitrite oxidizers, which convert nitrite to nitrate, and with heterotrophic microorganisms. The presence of nitrite oxidizers and heterotrophic bacteria has a positive influence on the growth of the ammonia oxidizers. Here, we present a study investigating the effect of nitrite oxidizers and heterotrophic bacteria on the proteome of a selected ammonia oxidizer in a defined culture to elucidate how these two groups improve the performance of the ammonia oxidizer. The results show that the presence of a nitrite oxidizer and heterotrophic bacteria reduced the stress for the ammonia oxidizer and resulted in more efficient energy generation. This study contributes to our understanding of microbe-microbe interactions, in particular between ammonia oxidizers and their neighboring microbial community.

Impact of methionine on a partial-nitritation biofilter.

It has been demonstrated that an anaerobic digestion process cannot attain an efficient removal of several amino acids, with methionine being one of the most persistent of these. Thus, the effect that methionine amino acid has over the partial-nitritation process with fixed-biofilm configuration in terms of performance and bacterial community dynamics has been investigated. With respect to the performance with no addition, 100 mg/L methionine loading decreased ammonium oxidation efficiency in 60% and 100% at concentrations of 300 and 500 mg/L methionine, respectively. Bacterial biomass sharply increased by 30, 65, and 230% with the addition of 100, 300, and 500 mg/L methionine, respectively. Bacterial community analysis showed that methionine addition supported the proliferation of a diversity of heterotrophic genera, such as Lysobacter and Micavibrio, and reduced the relative abundance of ammonium oxidizing genus Nitrosomonas. This research shows that the addition of methionine affects the performance of the partial-nitritation process. In this sense, amino acids can pose a threat for the of partial-nitritation process treating anaerobic digester supernatant at full-scale implementation.

Population dynamics of nitrifying bacteria for nitritation achieved in Johannesburg (JHB) process treating municipal wastewater.

Population dynamic of nitrifying bacteria was investigated for nitrogen removal from municipal wastewater. Nitritation was established with nitrite accumulation ratios above 85%. Quantitative PCR indicated that Nitrospira was dominant nitrite oxidizing bacteria (NOB) and Nitrobacter was few. During nitritation achieving, Nitrobacter was firstly eliminated, along with inhibition of Nitrospira bioactivities, then Nitrospira percentage declined and was finally washed out. Nitritation establishment depended on inhibiting and eliminating of NOB rather than ammonia oxidizing bacteria (AOB) enriching. This is the first study where population dynamics of Nitrobacter and Nitrospira were investigated to reveal mechanism of nitritation in a continuous-flow process. Phylogenetic analysis of AOB indicated that Nitrosomonas-like cluster and Nitrosomonas oligotropha were dominant AOB, accounting for 81.6% of amoA gene clone library. Community structure of AOB was similar to that of complete nitrification system with long hydraulic retention time, but different from that of nitritation reactor with low DO concentration.

Use of real-time QPCR in biokinetics and modeling of two different ammonia-oxidizing bacteria growing simultaneously.

A real-time quantitative polymerase chain reaction (QPCR) was used to evaluate biokinetic coefficients of Nitrosomonas nitrosa and N. cryotolerans clusters growing simultaneously in a batch mode of ammonia oxidation. The mathematical models based on Monod equation were employed to describe the competitive relationship between these clusters and were fitted to experimental data to obtain biokinetic values. The maximum growth rates (µ(m)), half-saturation coefficients (K(S)), microbial yields (Y) and decay coefficients (k(d)) of N. nitrosa and N. cryotolerans were 1.77 and 1.21 day(-1), 23.25 and 23.06 mg N·L(-1), 16 × 10(8) and 1 × 10(8) copies·mg N(-1), 0.26 and 0.20 day(-1), respectively. The estimated coefficients were applied for modeling continuous operations at various hydraulic retention times (HRTs) with an influent ammonia concentration of 300 mg N·L(-1). Modeling results revealed that ammonia oxidation efficiencies were achieved 55-98 % at 0.8-10 days HRTs and that the system was predicted to be washed out at HRT of 0.7 days. Overall, use of QPCR for estimating biokinetic coefficients of the two AOB cluster growing simultaneously by use of ammonia were successful. This idea may open a new direction towards biokinetics of ammonia oxidation in which respirometry tests are usually employed.

Analysis of microbial population dynamics in a partial nitrifying SBR at ambient temperature.

In this study, a lab-scale partial nitrifying sequencing batch reactor (SBR) was developed to investigate partial nitrification at ambient temperature (16-22 °C). Techniques of denaturing gradient gel electrophoresis (DGGE), cloning, and fluorescence in situ hybridization (FISH) were utilized simultaneously to study microbial population dynamics. Partial nitrification was effectively achieved in response to shifts of influent ammonium concentrations. DGGE results showed that higher ammonia concentration referred to lower ammonia-oxidizing bacteria (AOB) diversity in the SBR. Phylogenetic analysis revealed that all the predominant AOB was affiliated with Nitrosomonas genus. FISH analysis illustrated AOB was the predominant nitrifying bacteria of microbial compositions when SBR achieved partial nitrification (PN) at ambient temperature.

Dynamics of specific ammonia-oxidizing bacterial populations and nitrification in response to controlled shifts of ammonium concentrations in wastewater.

Ammonia-oxidizing bacteria (AOB) are essential for the nitrification process in wastewater treatment. To retain these slow-growing bacteria in wastewater treatment plants (WWTPs), they are often grown as biofilms, e.g., on nitrifying trickling filters (NTFs) or on carriers in moving bed biofilm reactors (MBBRs). On NTFs, a decreasing ammonium gradient is formed because of the AOB activity, resulting in low ammonium concentrations at the bottom and reduced biomass with depth. To optimize the NTF process, different ammonium feed strategies may be designed. This, however, requires knowledge about AOB population dynamics. Using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy, we followed biomass changes during 6 months, of three AOB populations on biofilm carriers. These were immersed in aerated MBBR tanks in a pilot plant receiving full-scale wastewater. Tanks were arranged in series, forming a wastewater ammonium gradient mimicking an NTF ammonium gradient. The biomass of one of the dominating Nitrosomonas oligotropha-like populations increased after an ammonium upshift, reaching levels comparable to the high ammonium control in 28 days, whereas a Nitrosomonas europaea-like population increased relatively slowly. The MBBR results, together with competition studies in NTF systems fed with wastewater under controlled ammonium regimes, suggest a differentiation between the two N. oligotropha populations, which may be important for WWTP nitrification.

Reactor performance in terms of COD and nitrogen removal and bacterial community structure of a three-stage rotating bioelectrochemical contactor.

Integrating microbial fuel cell (MFC) into rotating biological contactor (RBC) creates an opportunity for enhanced removal of COD and nitrogen coupled with energy generation from wastewater. In this study, a three-stage rotating bioelectrochemical contactor (referred to as RBC-MFC unit) integrating MFC with RBC technology was constructed for simultaneous removal of carbonaceous and nitrogenous compounds and electricity generation from a synthetic medium containing acetate and ammonium. The performance of the RBC-MFC unit was compared to a control reactor (referred to as RBC unit) that was operated under the same conditions but without current generation (i.e. open-circuit mode). The effect of hydraulic loading rate (HLR) and COD/N ratio on the performance of the two units was investigated. At low (3.05 gCOD g⁻¹N) and high COD/N ratio (6.64 gCOD g⁻¹N), both units achieved almost similar COD and ammonia-nitrogen removal. However, the RBC-MFC unit achieved significantly higher denitrification and nitrogen removal compared to the RBC unit indicating improved denitrification at the cathode due to current flow. The average voltage under 1000 Ω external resistance ranged between 0.03 and 0.30 V and between 0.02 and 0.21 V for stages 1 and 2 of the RBC-MFC unit. Pyrosequencing analysis of bacterial 16S rRNA gene revealed high bacterial diversity at the anode and cathode of both units. Genera that play a role in nitrification (Nitrospira; Nitrosomonas), denitrification (Comamonas; Thauera) and electricity generation (Geobacter) were identified at the electrodes. Geobacter was only detected on the anode of the RBC-MFC unit. Nitrifiers and denitrifiers were more abundant in the RBC-MFC unit compared to the RBC unit and were largely present on the cathode of both units suggesting that most of the nitrogen removal occurred at the cathode.

Correlating microbial community structure and composition with aeration intensity in submerged membrane bioreactors by 454 high-throughput pyrosequencing.

For understanding of the microbial community structure and composition under different aeration intensities, 454 high-throughput pyrosequencing was applied to analyze the 16S rRNA gene of bacteria in two submerged membrane bioreactors (MBRs) under low (R(L)) and high aeration (R(H)) conditions. In total, 7818 (R(L)) and 9353 (R(H)) high-quality reads were obtained, and 1230 (R(L)) and 924 (R(H)) operational taxonomic units (OTUs) were generated at 3% cutoff level, respectively. 454 pyrosequencing could also reveal the minority bacteria that were hardly detected by the conventional molecular methods. Although the core populations were shared with highly functional organization (>80%), clear differences between the samples in the two MBRs were revealed by richness-diversity indicators and Venn analyses. Notably, microbial diversity was decreased under high aeration condition, and the evolution of the populations was observed mainly in the shared OTUs. Moreover, specific comparison down to the class and genus level showed that the relative abundances of ß-Proteobacteria and γ-Proteobacteria in the R(H) community were respectively decreased by 41.5% and 66.6%, consistent with the observed membrane fouling mitigation during the reactor operation. It was also found that Nitrospira and Nitrosomonas, being nitrite oxidizing bacteria (NOB) and ammonium oxidizing bacteria (AOB), were the dominant phylogenetic groups at the genus level of both reactors, and that the high ratio of NOB to AOB populations well supported the complete ammonium oxidation performance in the two reactors. Although some populations of NOB and AOB decreased with the increase of aeration intensity, the functional stability of the nitrification process was less affected, probably due to the low influent substrate concentration and the high level of functional organization.

Effects of nitrogen dioxide and anoxia on global gene and protein expression in long-term continuous cultures of Nitrosomonas eutropha C91.

Nitrosomonas eutropha is an ammonia-oxidizing betaproteobacterium found in environments with high ammonium levels, such as wastewater treatment plants. The effects of NO(2) on gene and protein expression under oxic and anoxic conditions were determined by maintaining N. eutropha strain C91 in a chemostat fed with ammonium under oxic, oxic-plus-NO(2), and anoxic-plus-NO(2) culture conditions. Cells remained viable but ceased growing under anoxia; hence, the chemostat was switched from continuous to batch cultivation to retain biomass. After several weeks under each condition, biomass was harvested for total mRNA and protein isolation. Exposure of N. eutropha C91 to NO(2) under either oxic or anoxic conditions led to a decrease in proteins involved in N and C assimilation and storage and an increase in proteins involved in energy conservation, including ammonia monooxygenase (AmoCAB). Exposure to anoxia plus NO(2) resulted in increased representation of proteins and transcripts reflective of an energy-deprived state. Several proteins implicated in N-oxide metabolism were expressed and remained unchanged throughout the experiment, except for NorCB nitric oxide reductase, which was not detected in the proteome. Rather, NorY nitric oxide reductase was expressed under oxic-plus-NO(2) and anoxic-plus-NO(2) conditions. The results indicate that exposure to NO(2) results in an energy-deprived state of N. eutropha C91 and that anaerobic growth could not be supported with NO(2) as an oxidant.

Influence of growth manner on nitrifying bacterial communities and nitrification kinetics in three lab-scale bioreactors.

The effects of growth type, including attached growth, suspended growth, and combined growth, on the characteristics of communities of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were studied in three lab-scale Anaerobic/Anoxic(m)-Oxic(n) (AmOn) systems. These systems amplified activated sludge, biofilms, and a mixture of activated sludge and biofilm (AS-BF). Identical inocula were adopted to analyze the selective effects of mixed growth patterns on nitrifying bacteria. Fluctuations in the concentration of nitrifying bacteria over the 120 days of system operation were analyzed, as was the composition of nitrifying bacterial community in the stabilized stage. Analysis was conducted using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR. According to the DGGE patterns, the primary AOB lineages were Nitrosomonas europaea (six sequences), Nitrosomonas oligotropha (two sequences), and Nitrosospira (one sequence). The primary subclass of NOB community was Nitrospira, in which all identified sequences belonged to Nitrospira moscoviensis (14 sequences). Nitrobacter consisted of two lineages, namely Nitrobacter vulgaris (three sequences) and Nitrobacter alkalicus (two sequences). Under identical operating conditions, the composition of nitrifying bacterial communities in the AS-BF system demonstrated significant differences from those in the activated sludge system and those in the biofilm system. Major varieties included several new, dominant bacterial sequences in the AS-BF system, such as N. europaea and Nitrosospira and a higher concentration of AOB relative to the activated sludge system. However, no similar differences were discovered for the concentration of the NOB population. A kinetic study of nitrification demonstrated a higher maximum specific growth rate of mixed sludge and a lower half-saturation constant of mixed biofilm, indicating that the AS-BF system maintained relatively good nitrifying ability.