Search

Nitrospira is a common genus of nitrite-oxidising bacteria (NOB) found in wastewater treatment plants (WWTPs). To identify the key factors influencing the composition of NOB communities, research was conducted using both sequencing batch reactor (SBR) and continuous flow reactor under different conditions. High-throughput 16S rRNA gene sequencing revealed that Nitrospira (18.79% in R1 and 25.77% in R3) was the dominant NOB under low dissolved oxygen (DO) and low nitrite ( NO 2 - -N) concentrations, while Nitrobacter (21.26% in R2) was the dominant NOB under high DO and high NO 2 - -N concentrations. Flocculent and granule sludge were cultivated with Nitrospira as the dominant genus. Compared to Nitrospira flocculent sludge, Nitrospira granule sludge had higher inhibition threshold concentrations for free ammonia (FA) and free nitrous acid (FNA). It was more likely to resist adverse environmental disturbances. Furthermore, the effects of environmental factors such as temperature, pH, and DO on the activity of Nitrospira granular sludge were also studied. The results showed that the optimum temperature and pH for Nitrospira granular sludge were 36°C and 7.0, respectively. Additionally, Nitrospira granular sludge showed a higher dissolved oxygen half-saturation constant ( K o) of 3.67 ± 0.71 mg/L due to its morphological characteristics. However, the majority of WWTPs conditions do not meet the conditions for the Nitrospira granular sludge. Thus, it can be speculated that future development of aerobic partial nitrification granular sludge may automatically eliminate the influence of Nitrospira . This study provides a theoretical basis for a deeper understanding of Nitrospira and the development of future water treatment processes.

Wastewater with residual streptomycin sulphate usually contains high content of ammonia-nitrogen. However, the biological removal process of ammonia-nitrogen under streptomycin sulphate circumstance was unclear. In this study, short-term and long-term effects of streptomycin sulphate on biological nitrification systems, including AOB, NOB, SAOR, SNOR and SNPR, were evaluated comprehensively. The results indicated IC50 for AOB and NOB were 7.5 and 6.6 mg/L. SAOR and SNPR could be decreased to 3.43 ± 0.52 mg N/(g MLSS·h) and 0.24 ± 0.03 mg N/(g MLSS·h) while the addition of streptomycin sulphate was 10 mg/L. When streptomycin sulphate addition was stopped, nitrification ability recovered slightly, SAOR and SNPR increased to 9.37 ± 0.36 mg N/(g MLSS·h) and 1.66 ± 0.49 mg N/(g MLSS·h), respectively. The protein of EPS increased gradually during the acclimatization process, and the maximal protein value was 68.24 mg/g MLSS on the 100th day, however, no significant change of polysaccharose was observed during the acclimatization process. High abundance of ARGs and intI1 was detected in effluent and sludge of the biological treatment system. The maximal relative abundance of aadA1 in the sludge appeared on the 140th day, and increased by 0.99 orders of magnitude. Biological diversity decreased significantly during the acclimatization process, relative abundance of nitrosomonas was changed from 9.07% to 38.68% on the 61st day, while relative abundance of nitrobacter was changed from 1.30% to 0.64%. It should be noted that relative abundances of nitrosomonas and nitrobacter were reduced to 16.17% and 0.25% on the 140th day. This study would be helpful for nitrogen removal in wastewater with antibiotic.

Nitrite-oxidizing bacteria (NOB), which perform the second step of aerobic nitrification, play an important role in soil. In the present study, we report a novel isolate from agricultural soil affiliated with the genus Nitrobacter and its physiological characteristics. We sampled the surface soil of a vegetable field and obtained mixed culture A31 using the most probable number (MPN) method with inorganic medium containing 0.75‍ ‍mM urea (pH 5.5). The dilution-extinction procedure on culture A31 led to the isolation of a strain that was designated as Nitrobacter sp. A67. The nxrB1 gene sequence of Nitrobacter sp. A67 (302 bp) was classified into Cluster 5, and the highest sequence identity was 96.10% with Nitrobacter sp. BS5/19. The NO 2 - oxidation activity of Nitrobacter sp. A67 was investigated at various pH. The optimum pH for NO 2 - oxidation was 5.8-6.4. This result indicates that Nitrobacter sp. A67 is a moderately acidophilic nitrite-oxidizing bacterium.

The effect of nanoplastics (NPs) on nitrite oxidation bacteria (NOB) community in treating high-strength wastewater remains unclear, which seriously affects the stability of nitrogen removal process. In this study, highly active nitrifying sludge was enriched and exposed to 50 nm polystyrene NPs (PS-NPs) for short-term (1, 100, 500, and 1000 mg/L, 1.5 hr) and long-term (1, 10, 100 mg/L, 40 days) at high nitrite concentration. In contrast to previous studies, our results showed that the exposures to PS-NPs had little effect on nitrifying performances. After long-term exposure, the protein/polysaccharide ratios in extracellular polymeric substances (EPS) were positively correlated with PS-NPs concentrations (0.78-0.99). The produced reactive oxygen species (ROS) were gradually removed, and PS-NPs higher than 10 mg/L caused damage to membrane integrity. Long-term exposure for 40 days increased the community diversity and caused significant differences between the control and exposed communities. The control group were dominated by Nitrobacter and Exiguobacterium, while the exposure group was dominated by Bacillus, Mycobacterium, and Nitrospira. A noticeable shift in the NOB community from Nitrobacter (26.5% to 3.4%) to Nitrospira (1.61% to 14.27%) was observed. A KEGG analysis indicated a decrease in cell growth and death, cell motility and energy metabolism. It appeared that NOB could adapt to PS-NPs stress through enhanced secretion and removal of oxidative damage. Overall, this study provided new insights into the response mechanism of NOB to PS-NPs exposure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2023. Published by Elsevier B.V.)

Toxicological batch assays are essential to assess a compound's acute effect on microorganisms. This methodology is frequently employed to evaluate the effect of contaminants in sensitive microbial communities from wastewater treatment plants (WWTPs), such as autotrophic nitrifying populations. However, despite nitrifying batch assays being commonly mentioned in the literature, their experimental design criteria are rarely reported or overlooked. Here, we found that slight deviations in culture preparations and conditions impacted bacterial community performance and could skew assay results. From pre-experimental trials and experience, we determined how mishandling and treatment of cultures could affect nitrification activity. While media and biomass preparations are needed to establish baseline conditions (e.g., biomass washing), we found extensive centrifugation selectively destabilised nitrification activities. Further, it is paramount that the air supply is adjusted to minimise nitrite build-up in the culture and maintain suitable aeration levels without sparging ammonia. DMSO and acetone up to 0.03% (v/v) were suitable organic solvents with minimal impact on nitrification activity. In the nitrification assays with allylthiourea (ATU), dilute cultures exhibited more significant inhibition than concentrated cultures. So there were biomass-related effects; however, these differences minimally impacted the EC 50 values. Using different nutrient-media compositions had a minimal effect; however, switching mineral media for the toxicity test from the original cultivation media is not recommended because it reduced the original biomass nitrification capacity. Our results demonstrated that these factors substantially impact the performance of the nitrifying inoculum used in acute bioassays, and consequently, affect the response of AOB-NOB populations during the toxicant exposure. These are not highlighted in operation standards, and unfortunately, they can have significant consequential impacts on the determinations of toxicological endpoints. Moreover, the practical procedures tested here could support other authors in developing testing methodologies, adding quality checks in the experimental framework with minimal waste of time and resources., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Nitrification is a key step in biological nitrogen transformation which depends on the performance of specialized microorganisms. Generally, nitrifying bacteria present a low growth rate and performance which can be improved when immobilized as a biofilm. The development of new materials suitable for the immobilization of nitrifying microorganisms is very important in nitrification and wastewater treatment. In this study, the effect of eggshell powder on biofilm formation by Nitrosomonas europaea an ammonium-oxidizing bacteria and Nitrobacter vulgaris a nitrite-oxidizing bacteria, on new polymeric supports were analyzed. Polylactic acid, polyvinyl chloride and polystyrene were tested to produce polymer-eggshells powder composites and used as biofilm supports for nitrifying bacteria. The support material was characterized to identify the most suitable polymer-eggshells powder combination for the cell adhesion and biofilm formation. The nitrification results showed a highest nitrate production of 42 mg NO 3 - -N/L with polylactic acid-eggshell composite, with the best surface properties for cellular adhesion. Finally, scanning electron microscopy micrographs confirmed the best biofilm formed on polylactic acid-eggshell., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Bacterial hopanoid lipids are ubiquitous in the geologic record and serve as biomarkers for reconstructing Earth's climatic and biogeochemical evolution. Specifically, the abundance of 2-methylhopanoids deposited during Mesozoic ocean anoxic events (OAEs) and other intervals has been interpreted to reflect proliferation of nitrogen-fixing marine cyanobacteria. However, there currently is no conclusive evidence for 2-methylhopanoid production by extant marine cyanobacteria. As an alternative explanation, here we report 2-methylhopanoid production by bacteria of the genus Nitrobacter , cosmopolitan nitrite oxidizers that inhabit nutrient-rich freshwater, brackish, and marine environments. The model organism Nitrobacter vulgaris produced only trace amounts of 2-methylhopanoids when grown in minimal medium or with added methionine, the presumed biosynthetic methyl donor. Supplementation of cultures with cobalamin (vitamin B 12 ) increased nitrite oxidation rates and stimulated a 33-fold increase of 2-methylhopanoid abundance, indicating that the biosynthetic reaction mechanism is cobalamin dependent. Because Nitrobacter spp. cannot synthesize cobalamin, we postulate that they acquire it from organisms inhabiting a shared ecological niche-for example, ammonia-oxidizing archaea. We propose that during nutrient-rich conditions, cobalamin-based mutualism intensifies upper water column nitrification, thus promoting 2-methylhopanoid deposition. In contrast, anoxia underlying oligotrophic surface ocean conditions in restricted basins would prompt shoaling of anaerobic ammonium oxidation, leading to low observed 2-methylhopanoid abundances. The first scenario is consistent with hypotheses of enhanced nutrient loading during OAEs, while the second is consistent with the sedimentary record of Pliocene-Pleistocene Mediterranean sapropel events. We thus hypothesize that nitrogen cycling in the Pliocene-Pleistocene Mediterranean resembled modern, highly stratified basins, whereas no modern analog exists for OAEs., Competing Interests: The authors declare no competing interest.

Approximately two percent of the world's energy is consumed in the production of ammonia from hydrogen and nitrogen gas. Ammonia is used as a fertilizer ingredient for agriculture and distributed in the environment on an enormous scale to promote crop growth in intensive farming. Only 30-50 % of the nitrogen applied is assimilated by crop plants; the remaining 50-70 % goes into biological processes such as nitrification by microbial metabolism in the soil. This leads to an imbalance in the global nitrogen cycle and higher nitrous oxide emissions (a potent and significant greenhouse gas) as well as contamination of ground and surface waters by nitrate from the nitrogen-fertilized farmland. This Review gives a critical overview of the current knowledge of soil microbes involved in the chemistry of ammonia nitrification, the structures and mechanisms of the enzymes involved, and phytochemicals capable of inhibiting ammonia nitrification., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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±10mgNH 4 + -NL -1 d -1 removal rate. In reactor 2, the nitrifiers and ureolytic heterotrophs were fed with urine and achieved a 15±6mg NO 3 - -NL -1 d -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 -1 d -1 . Organics removal in the reactor (69±15%) should be optimized to generate a nitrate fertilizer for future space applications., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Nitrobacter winogradskyi is an abundant, intensively studied autotrophic nitrite-oxidizing bacterium, which is frequently used as a model strain in the two-step nitrification of ammonia (NH 3 ) to nitrate (NO 3 - ) via nitrite (NO 2 - ), either in activated sludge, agricultural field studies or more recently in artificial microbial consortia for organic hydroponics. We observed a hitherto unknown cobalt ion-dependent inhibition of cell growth and NO 2 - oxidation activity of N. winogradskyi in a mineral medium, which strongly depended on accompanying Ca 2+ and Mg 2+ concentrations. This inhibition was bacteriostatic, but susceptible to natural chelators. l-Histidine effectively restored cell growth and NO 2 - oxidation activity of N. winogradskyi in mineral media containing Co 2+ with >90% recovery. Our results suggest that Co 2+ competed with alkaline earth metals during uptake and that its toxicity was significantly reduced by complexation., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

This study investigated the initiation and maintenance of nitritation in a membrane bioreactor (MBR) with long solids retention time (SRT) of 43.8 days. Nitritation was initiated within 65 days in the MBR via dissolved oxygen (DO) limitation (<0.5 mg/L). However, nitrite oxidizing bacteria (NOB) (Nitrospira and Nitrobacter) acclimated to the low DO environment and proliferated from day 81, leading to nitrate accumulation. Thereafter, the combined strategy of DO limitation and in-situ generated free nitrous acid (FNA) shock successfully restored and maintained stable nitritation for >70 days. Quantitative polymerase chain reaction (qPCR) results showed that cell abundances of Nitrospira and Nitrobacter decreased by between 50.0 to 68.9% and 60.6 to 96.4%, respectively following the FNA shocks. The maximum ammonium loading rate achieved was 1.81 kg N/(m 3  day) with ammonium removal ratio and nitrite accumulation ratio of over 0.97 and 0.96, respectively. Average emission rate of N 2 O from the MBR was 2.1 ± 0.72% of ammonium removed. FNA shock on day 195 reduced the N 2 O emission by 13.6%. The strategy developed in this study verified that spiked FNA shock together with DO limitation can be used for maintaining nitritation in MBRs with long SRTs. This method can potentially allow for maintaining nitritation at relatively low capital and operating expenditure when treating high concentration ammonium wastewater., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A sequencing batch reactor (SBR) was operated in this study to investigate the effect of temperature on the kinetics of Nitrobacter activity among nitrite oxidizing bacteria. At the beginning of the experiment, the NO 2 - -N concentration in the influent was changed to enrich Nitrobacter . Then, the sludge with enriched Nitrobacter was employed to determine the variation of the specific nitrite oxidation rate (SNiOR) during the nitrite oxidation process in batch tests. Metagenomics species annotation and abundance analysis showed that Nitrobacter accounted for 40.3% of the total bacterial population. The variation of SNiOR in the nitrite oxidation process was investigated under different NO 2 - -N concentrations. The effect of temperature on the kinetics of Nitrobacter was investigated using the Monod model. Furthermore, the kinetics model of the effect of temperature on Nitrobacter activity was fitted for statistical analysis. The results showed that SNiOR reached its maximum at 30℃, which was 1.31 g·(g·d) -1 . Statistical analysis showed that the Monod equation could describe the effect of substrate concentration on Nitrobacter activity under different temperature conditions. Calculating the temperature coefficient ( θ ) in different temperature intervals based on the Phelps equation, showed that when the system temperature is lower than 25℃ or higher than 30℃, the reaction rate is more sensitive to temperature changes.

Although Nitrobacter winogradskyi is an important chemoorganotrophic organism for the study of nitrite-oxidizing bacteria physiology as well as nitrification, until now, the mixotrophic medium for this organism growth has not been optimized, comprehensively. In this study, we aimed to improve the growth medium of N. winogradskyi using the one-factor-at-a-time (NaNO 2 , glycerol, pH) method. In addition, a further experimental design was carried out based on central composite design with response surface methodology. Different combinations of the three cultural parameters were fitted by multiple regression analysis to calculate the predicted response. Our results suggest that optimal culture condition for the growth of N. winogradskyi was a modified DSMZ 756a medium containing NaNO 2 (5·74 g l -1 ) and glycerol (37·88 mmol l -1 ), pH 7·83, a temperature of 28°C and agitation at 120 rev min -1 . The results from a validation experiment (bacterial growth: OD 600 1·0293) were close to the value predicted by the quadratic model (OD 600 1·0994). In addition, we uncovered the potential mechanism at the cellular and ultrastructural levels. The results indicated that glycerol in the media enhanced the rate of cell division and cell growth by increasing the accumulation of polyphosphates and phosphorus, and high concentrations of NaNO 2 provided sufficient energy for growth and contributed to the generation of carboxysomes in cells for CO 2 fixation., Significance and Impact of the Study: Due to the extremely slow growth rate and the low growth yield of ammonia-oxidizing bacteria and NOB (nitrite-oxidizing bacteria), nitrification is still the rate-limiting step of nitrogen cycle in the current research. Nitrobacter winogradskyi, an important NOB, participates in the second step of nitrification in water and soil. This study reported an optimized culture condition for N. winogradskyi, which increased the growth yield by 5·06 times than that in the basal medium and uncovered the potential mechanism. We expect our study will contribute to the research on water and soil nitrogen cycle. In addition, the optimized culture conditions have the potential to be suitable for the chemoorganotrophic growth of other nitrifiers., (© 2018 The Society for Applied Microbiology.)

This work studied the microbial community in partial nitritation and complete nitrification processes, which were applied to treat the low Carbon Nitrogen ratio wastewater. The phospholipid fatty acid and quantitative PCR analysis showed that the sludge circulating ratio of 75% resulted in a good microbial growth and a higher abundance of ammonia oxidizing bacteria relative to the nitrite oxidizing bacteria. The Betaproteobacteria were observed to compose the most abundant sludge bacterial groups in the two processes, based on phylogenetic analysis. The phylogenetic analysis of both 16S rRNA and amoA gene indicated that the Nitrosomonas sp. were the dominant ammonia oxidizing bacteria in the partial nitritation process. The relative abundance of nitrite oxidizing bacteria, such as Nitrobacter sp. and Nitrospira sp., were significantly lower in the partial nitritation system over the complete nitrification system. The abundance of Planctomycetes was higher in the partial nitritation process, indicating the anammox reaction occurred in the partial nitritation system. These results suggested the nitrite accumulation rate of circulating ratios 75% was the highest, with an average of 92%,and a possibility to treat the low Carbon Nitrogen ratio wastewater using the partial nitritation/anammox process.

In this study, the effects of the ammonium loading rate (ALR) and inorganic carbon loading rate (ILR) on the nitrification performance and composition of a nitrifying bacterial community were investigated in a moving bed biofilm reactor, using poly(vinyl alcohol) (PVA) sponge cubes as a supporting carrier. Between the two ALRs of 0.36 and 2.16 kg-N m -1 d -1 , stable partial nitritation was achieved at the higher ALR. Inorganic carbon was dosed at high levels: 33.1, 22.0, 16.4, 11.0, and 5.4 times the theoretical amount. Nonetheless, nitrification efficiency was not affected by the ILR at the two ALRs. Quantitative PCR analysis of ammonia- and nitrite-oxidizing bacteria revealed that ALR is an important determinant of partial nitritation by accumulating ammonia-oxidizing bacteria in the nitrification system. In comparison, two nitrite-oxidizing bacterial genera (Nitrobacter and Nitrospira) showed almost the same relative abundance at various ALRs and ILRs. Terminal restriction fragment length polymorphism targeting the gene of ammonia monooxygenase subunit A revealed that Nitrosomonas europaea dominated under all conditions.

Biocomposites, i.e., materials consisting of metabolically active microorganisms embedded in a synthetic extracellular matrix, may find applications as highly specific catalysts in bioproduction and bioremediation. 3D constructs based on fibrous biocomposites, so-called "artificial biofilms," are of particular interest in this context. The inability to produce biocomposite fibers of sufficient mechanical strength for processing into bioactive fabrics has so far hindered progress in the area. Herein a method is proposed for the direct wet spinning of microfibers suitable for weaving and knitting. Metabolically active bacteria (either Shewanella oneidensis or Nitrobacter winogradskyi (N. winogradskyi)) are embedded in these fibers, using poly(vinyl alcohol) as matrix. The produced microfibers have a partially crystalline structure and are stable in water without further treatment, such as coating. In a first application, their potential for nitrite removal (N. winogradskyi) is demonstrated, a typical challenge in potable water treatment., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The factors influencing how soil nitrite (NO2-)- and ammonia (NH3)-oxidizing activities remain coupled are unknown. A short-term study (<48 h) was conducted to examine the dynamics of NO2--oxidizing activity and the accumulation of NO2- in three Oregon soils stimulated by the addition of 1 mM NH4+ in soil slurry. Nitrite initially accumulated in all three soils; its subsequent decline or slowing of the accumulation of the NO2- pool by 24 h was accompanied by an increase in the size of the nitrate (NO3-) pool, indicating a change in NO2- oxidation kinetics. Bacterial protein synthesis inhibitors prevented the NO2- pool decline, resulting in a larger accumulation in all three soils. Although no significant increases in NO2--oxidizing bacteria nxrA (Nitrobacter) and nxrB (Nitrospira) gene abundances were detected over the time course, maximum NO2- consumption rates increased 2-fold in the treatment without antibiotics compared to no change with antibiotics. No changes were observed in the apparent half saturation constant (Km) values for NO2- consumption. This study demonstrates phenotypic flexibility among soil NO2- oxidizers, which can undergo protein synthesis-dependent increases in NO2- consumption rates to match NH3 oxidation rates and recouple nitrification.

The effect of inorganic suspended solids (ISS) on nitrification in freshwater samples has been described inconsistently and remains unclear. This study therefore investigated the effects of the chemical characteristics and concentration of ISS on the nitrification rate by focusing on Nitrosomonas europaea and Nitrobacter winogradskyi as the two most dominant nitrification species in freshwater. Batch-wise experiments were conducted using three chemically well-characterized ISS (i.e. the clay minerals montmorillonite, sericite, and kaolinite in the concentration range 0-1,000 mg L -1 ). The results show that the ammonium oxidation rate constant (k NH4 ) was significantly affected by the ISS type, whereas changes in the ISS concentration had an insignificant effect on k NH4 , except for kaolinite. The highest k NH4 was observed in samples containing sericite (k NH4 , 0.067 L mg -1 day -1 ), followed by samples containing montmorillonite (k NH4 , 0.044 L mg -1 day -1 ). The ammonium oxidation rate was low in the control and kaolinite samples. Nitrite oxidation was enhanced in the presence of all types of ISS. The rate constants of ISS-mediated nitrite oxidation (k NO2 , 0.13-0.21 L mg -1 day -1 ) were not significantly different among the three types of ISS, but k NO2 was significantly affected by ISS concentration. Overall, our study indicated various effects of the ISS type and concentration on nitrification and, in particular, a notable positive effect of sericite.

Nitrite oxidation is recognized as an essential process of biogeochemical nitrogen cycling in agricultural ecosystems. How nitrite-oxidizing bacteria (NOB) respond to land managements (the effect from the long-term straw incorporation and environmental variability caused by the shift from the upland stage to the paddy stage) in a rapeseed-rice rotation field remains unclear. We found the nitrite oxidation (NO) in soils increased from the upland stage to the paddy stage. An inhibitory effect of the long-term straw incorporation on NO was detectable in the upland stage. The abundance of Nitrospira was always greater than Nitrobacter, and it was affected by the rice-growing and straw incorporation while Nitrobacter was not. NO correlated positively with the abundance of Nitrospira and with soluble sulfate (SO 4 2- ), soil moisture, pH and NH 4 + . The high-throughput sequencing analysis of the nitrite oxidoreductase nxrA and nxrB genes for Nitrobacter- and Nitrospira-like NOB was performed respectively. The dominating (relative abundance>1%) operational taxonomic units (OTUs) from Nitrobacter were closely related to Nitrobacter hamburgensis, whereas those from Nitrospira were affiliated with or related to lineage II, lineage V and several unknown groups. Heatmap analysis showed that a few dominant Nitrobacter OTUs were affected by the straw treatment or the rice-growing, and half of the dominant Nitrospira ones were explained by at least one of the variables. Multi-response permutation procedure (MRPP) and redundancy analyses showed that the Nitrospira-like NOB community changes were significantly shaped by the land managements and the soil chemical properties, including pH, moisture and NH 4 + , whereas that of the Nitrobacter-like NOB community was not. These results suggested that Nitrospira are more sensitive than Nitrobacter to land management in acid and fertilized soils of a rapeseed-rice rotation field trial., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The conditions present in both in vitro and in vivo ecosystems determine the microbial population harbouring it. One commonly accepted theory is that a species with a high substrate affinity and low growth rate (k-strategist) will win the competition against a second species with a lower substrate affinity and higher growth rate (r-strategist) if both species are subjected to low substrate concentrations. In this study two nitrite oxidizing bacteria (NOB), Nitrospira defluvii (k-strategist) and Nitrobacter vulgaris (r-strategist), were cultivated in a continuous reactor systems. The minimal hydraulic retention time (HRT) required for maintaining the slower growing Nitrospira was first determined. A reactor containing Nitrobacter was set to the same HRT and Nitrospira was injected to evaluate the effect of the dilution rate on the competition between both species. By following the microbial population dynamics with qPCR analysis, it was shown that not only the substrate affinity drives the competition between k- and r-strategists but also the dilution rate. Experimental data and numerical simulations both revealed that the washout of Nitrobacter was significantly delayed at dilution rates close to the μmax of Nitrospira. The competition could be even reverted towards Nitrobacter (r-strategist) despite of low nitrite concentrations and dilution rates lower than the μmax of Nitrospira.

The role of autotrophic nitrification on the biodegradation of toxic organic micro-pollutants presented in landfill leachate was assessed. A two-stage MBR system consisting of an inclined tube incorporated anoxic reactor followed by aerobic submerged membrane reactor was operated under long sludge age condition in which nitrifying bacteria could be enriched. During the reactor operation, organic removal efficiencies were more than 90% whereas phenolic compounds including bisphenol A (BPA) and 4-methyl-2,6-di-tert-butylphenol (BHT) were removed by 65 and 70% mainly through biodegradation in the aerobic reactor even at high feed concentrations of 1000μg/L for both compounds. Batch experiments revealed that enriched nitrifying sludge with nitrifying activities could biodegraded 88 and 75% of BPA and BHT, largely improved from non-nitrifying sludge and enriched nitrifying sludge with the presence of inhibitor. The first-order kinetic rates of BHT and BPA removal were 0.0108 and 0.096h -1 , also enhanced by 44% from the non-nitrifying sludge., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The aim of this work was to study co-cultivation of nitrifiers with microalgae as a non-intrusive technique for selective removal of oxygen generated by microalgae. Biomass concentration was, at least, 23% higher in mixed-cultures where nitrifiers kept the dissolved oxygen concentration below 9.0μLL(-1) than in control Chlorella vulgaris axenic-cultures where the concentration of dissolved oxygen was higher than 10.0μLL(-1). This approach to eliminating oxygen inhibition of microalgal growth could become the basis for the development of advanced microalgae reactors for removal of CO2 from the atmosphere, and concentrated CO2 streams. CO2 sequestration would become a chemically and geologically safer and environmentally more sound technology provided it uses microalgal, or other biomass, instead of CO2, for carbon storage., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Nitrite-oxidizing bacteria (NOB) catalyze the second step of nitrification, nitrite oxidation to nitrate, which is an important process of the biogeochemical nitrogen cycle. NOB were traditionally perceived as physiologically restricted organisms and were less intensively studied than other nitrogen-cycling microorganisms. This picture is in contrast to new discoveries of an unexpected high diversity of mostly uncultured NOB and a great physiological versatility, which includes complex microbe-microbe interactions and lifestyles outside the nitrogen cycle. Most surprisingly, close relatives to NOB perform complete nitrification (ammonia oxidation to nitrate) and this finding will have far-reaching implications for nitrification research. We review recent work that has changed our perspective on NOB and provides a new basis for future studies on these enigmatic organisms., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Urine contains the majority of nutrients in urban wastewaters and is an ideal nutrient recovery target. In this study, stabilization of real undiluted urine through nitrification and subsequent microalgae cultivation were explored as strategy for biological nutrient recovery. A nitrifying inoculum screening revealed a commercial aquaculture inoculum to have the highest halotolerance. This inoculum was compared with municipal activated sludge for the start-up of two nitrification membrane bioreactors. Complete nitrification of undiluted urine was achieved in both systems at a conductivity of 75mScm(-1) and loading rate above 450mgNL(-1)d(-1). The halotolerant inoculum shortened the start-up time with 54%. Nitrite oxidizers showed faster salt adaptation and Nitrobacter spp. became the dominant nitrite oxidizers. Nitrified urine as growth medium for Arthrospira platensis demonstrated superior growth compared to untreated urine and resulted in a high protein content of 62%. This two-stage strategy is therefore a promising approach for biological nutrient recovery., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Over the coming decades nitrous oxide (N2O) is expected to become a dominant greenhouse gas and atmospheric ozone depleting substance. In wastewater treatment systems, N2O is majorly produced by nitrifying microbes through biochemical reduction of nitrite (NO2(-)) and nitric oxide (NO). However it is unknown if the amount of N2O formed is affected by alternative NO redox reactions catalyzed by oxidative nitrite oxidoreductase (NirK), cytochromes (i.e., P460 [CytP460] and 554 [Cyt554 ]) and flavohemoglobins (Hmp) in ammonia- and nitrite-oxidizing bacteria (AOB and NOB, respectively). In this study, a mathematical model is developed to assess how N2O formation is affected by such alternative nitrogen redox transformations. The developed multispecies metabolic network model captures the nitrogen respiratory pathways inferred from genomes of eight AOB and NOB species. The performance of model variants, obtained as different combinations of active NO redox reactions, was assessed against nine experimental datasets for nitrifying cultures producing N2O at different concentration of electron donor and acceptor. Model predicted metabolic fluxes show that only variants that included NO oxidation to NO2(-) by CytP460 and Hmp in AOB gave statistically similar estimates to observed production rates of N2O, NO, NO2(-) and nitrate (NO3(-)), together with fractions of AOB and NOB species in biomass. Simulations showed that NO oxidation to NO2(-) decreased N2O formation by 60% without changing culture's NO2(-) production rate. Model variants including NO reduction to N2O by Cyt554 and cNor in NOB did not improve the accuracy of experimental datasets estimates, suggesting null N2O production by NOB during nitrification. Finally, the analysis shows that in nitrifying cultures transitioning from dissolved oxygen levels above 3.8 ± 0.38 to <1.5 ± 0.8 mg/L, NOB cells can oxidize the NO produced by AOB through reactions catalyzed by oxidative NirK., (© 2015 Wiley Periodicals, Inc.)

It is crucial to reveal the regulatory mechanism of nitrification to understand nitrogen conversion in agricultural systems and wastewater treatment. In this study, the nwiI gene of Nitrobacter winogradskyi was confirmed to be a homoserine lactone synthase by heterologous expression in Escherichia coli that synthesized several acyl-homoserine lactone signals with 7 to 11 carbon acyl groups. A novel signal, 7, 8-trans-N-(decanoyl) homoserine lactone (C10:1-HSL), was identified in both N. winogradskyi and the recombined E. coli. Furthermore, this novel signal also triggered variances in the nitrification rate and the level of transcripts for the genes involved in the nitrification process. These results indicate that quorum sensing may have a potential role in regulating nitrogen metabolism.

Microbial communities have a key role for the performance of engineered ecosystems such as waste gas biofilters. Maintaining constant performance despite fluctuating environmental conditions is of prime interest, but it is highly challenging because the mechanisms that drive the response of microbial communities to disturbances still have to be disentangled. Here we demonstrate that the bioprocess performance and stability can be improved and reinforced in the face of disturbances, through a rationally predefined strategy of microbial resource management (MRM). This strategy was experimentally validated in replicated pilot-scale nitrifying gas-biofilters, for the two steps of nitrification. The associated biological mechanisms were unraveled through analysis of functions, abundances and community compositions for the major actors of nitrification in these biofilters, that is, ammonia-oxidizing bacteria (AOB) and Nitrobacter-like nitrite-oxidizers (NOB). Our MRM strategy, based on the application of successive, transient perturbations of increasing intensity, enabled to steer the nitrifier community in a favorable way through the selection of more resistant AOB and NOB sharing functional gene sequences close to those of, respectively, Nitrosomonas eutropha and Nitrobacter hamburgensis that are well adapted to high N load. The induced community shifts resulted in significant enhancement of nitrification resilience capacity following the intense perturbation.

Oxygen is considered as a limiting factor for nitrification in rice paddy soil. However, little is known about how the nitrifying microbial community responds to different oxygen concentrations at community and transcript level. In this study, soil and roots were harvested from 50-day-old rice microcosms and were incubated for up to 45 days under two oxygen concentrations: 2 % O(2) and 20 % O(2) (ambient air). Nitrification rates were measured from the accumulation of nitrite plus nitrate. The population dynamics of bacterial (AOB) and archaeal (AOA) ammonia oxidizers was determined from the abundance (using quantitative PCR (qPCR)) and composition (using terminal restriction fragment length polymorphism and cloning/sequencing) of their amoA genes, that of nitrite oxidizers (NOB) by quantifying the nxrA gene of Nitrobacter spp. and the 16S rRNA gene of Nitrospira spp. The activity of the nitrifiers was determined by quantifying the copy numbers of amoA and nxrA transcripts (using RT-qPCR). Different oxygen concentrations did not affect the community compositions of AOB, AOA, and NOB, which however were different between surface soil, bottom soil, and rice roots. However, nitrification rates were higher under ambient air than 2 % O(2), and abundance and transcript activities of AOB, but not of AOA, were also higher. Abundance and transcript copy numbers of Nitrobacter were also higher at ambient air. These results indicate that AOB and NOB, but not AOA, were sensitive to oxygen availability.

In aerobic granular sludge (AGS), the growth of nitrite oxidizing bacteria (NOB) can be uncoupled from the nitrite supply of ammonia oxidizing bacteria (AOB). Besides, unlike for conventional activated sludge, Nitrobacter was found to be the dominant NOB and not Nitrospira. To explain these experimental observations, two possible pathways have been put forward in literature. The first one involves the availability of additional nitrite from partial denitrification (nitrite-loop) and the second one consists of mixotrophic growth of Nitrobacter in the presence of acetate (ping-pong). In this contribution, mathematical models were set up to assess the possibility of these pathways to explain the reported observations. Simulation results revealed that both pathways influenced the nitrifier distribution in the granules. The nitrite-loop pathway led to an elevated NOB/AOB ratio, while mixotrophic growth of Nitrobacter guaranteed their predominance among the NOB population. Besides, mixotrophic growth of Nitrobacter could lead to NO emission from AGS. An increasing temperature and/or a decreasing oxygen concentration led to an elevated NOB/AOB ratio and increased NO emissions.

In this study, the impact of COD/N ratio and feeding regime on the dynamics of heterotrophs and nitrifiers in moving-bed biofilm reactors was addressed. Based on DGGE analysis of 16S rRNA genes, the influent COD was found to be the main factor determining the overall bacterial diversity. The amoA-gene-based analysis suggested that the dynamic behavior of the substrate in continuous and pulse-feeding reactors influenced the selection of specific ammonium-oxidizing bacteria (AOB) strains. Furthermore, AOB diversity was directly related to the applied COD/N ratio and ammonium-nitrogen load. Maximum specific ammonium oxidation rates observed under non-substrate-limiting conditions were observed to be proportional to the fraction of nitrifiers within the bacterial community. FISH analysis revealed that Nitrosomonas genus dominated the AOB community in all reactors. Moreover, Nitrospira was found to be the only nitrite-oxidizing bacteria (NOB) in the fully autotrophic system, whereas Nitrobacter represented the dominant NOB genus in the organic carbon-fed reactors., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Nitrobacter winogradskyi is a chemolithotrophic bacterium that plays a role in the nitrogen cycle by oxidizing nitrite to nitrate. Here, we demonstrate a functional N-acyl-homoserine lactone (acyl-HSL) synthase in this bacterium. The N. winogradskyi genome contains genes encoding a putative acyl-HSL autoinducer synthase (nwi0626, nwiI) and a putative acyl-HSL autoinducer receptor (nwi0627, nwiR) with amino acid sequences 38 to 78% identical to those in Rhodopseudomonas palustris and other Rhizobiales. Expression of nwiI and nwiR correlated with acyl-HSL production during culture. N. winogradskyi produces two distinct acyl-HSLs, N-decanoyl-l-homoserine lactone (C10-HSL) and a monounsaturated acyl-HSL (C10:1-HSL), in a cell-density- and growth phase-dependent manner, during batch and chemostat culture. The acyl-HSLs were detected by bioassay and identified by ultraperformance liquid chromatography with information-dependent acquisition mass spectrometry (UPLC-IDA-MS). The C=C bond in C10:1-HSL was confirmed by conversion into bromohydrin and detection by UPLC-IDA-MS., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Partial nitritation for a low-strength wastewater at low temperature was stably achieved in an aerobic granular reactor. A bench-scale granular sludge bioreactor was operated in continuous mode treating an influent of 70 mg N-NH4(+) L(-1) to mimic pretreated municipal nitrogenous wastewater and the temperature was progressively decreased from 30 to 12.5 °C. A suitable effluent nitrite to ammonium concentrations ratio to a subsequent anammox reactor was maintained stable during 300 days at 12.5 °C. The average applied nitrogen loading rate at 12.5 °C was 0.7 ± 0.3 g N L(-1) d(-1), with an effluent nitrate concentration of only 2.5 ± 0.7 mg N-NO3(-) L(-1). The biomass fraction of nitrite-oxidizing bacteria (NOB) in the granular sludge decreased from 19% to only 1% in 6 months of reactor operation at 12.5 °C. Nitrobacter spp. where found as the dominant NOB population, whereas Nitrospira spp. were not detected. Simulations indicated that: (i) NOB would only be effectively repressed when their oxygen half-saturation coefficient was higher than that of ammonia-oxidizing bacteria; and (ii) a lower specific growth rate of NOB was maintained at any point in the biofilm (even at 12.5 °C) due to the bulk ammonium concentration imposed through the control strategy., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Herein, we report on composite materials of biologically active microorganisms placed in a synthetic polymer matrix. These so-called "living composites" were utilized for gold sequestration (Micrococcus luteus) and bioremediation of nitrite (Nitrobacter winogradskyi) to demonstrate functionality. For the preparation of the living composites the bacteria were first encased in a water-soluble polymer fiber (poly(vinyl alcohol), PVA) followed by coating the fibers with a shell of hydrophobic poly(p-xylylene) (PPX) by chemical vapor deposition (CVD). The combination of bacteria with polymer materials assured the stability and biologically activity of the bacteria in an aqueous environment for several weeks., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Free ammonia (FA) inhibition on nitrite-oxidized bacteria (NOB) and real-time control are used to achieve nitrogen removal from landfill leachate via nitrite pathway at low temperatures in sequencing batch reactor. The inhibition of FA on NOB activity during the aerobic period was prolonged using real-time control. The degree of nitrite accumulation was monitored along with variations of the ammonia-oxidizing bacteria and NOB population using fluorescence in situ hybridization techniques. It is demonstrated that the end-point of ammonia oxidization is detected from the on-line measured dissolved oxygen, oxidization-reduction potential, and pH signals, which could avoid the loss the FA inhibition on NOB caused by excess aeration. At low temperature (13.0-17.6°C), the level of nitrite pathway rapidly increased from 19.8% to 90%, suggesting that nitritation was successfully started up at low temperature by applying syntrophic association of the FA inhibition and real-time control, and then this high level of nitrite pathway was stably maintained for as long as 233 days. Mechanism analysis shows that the establishment of nitritation was primarily the result of predominant ammonia-oxidizing bacteria developed in the nitrifying bacteria population compared to NOB. This was mainly due to a gradual reduction of nitrite amount that is available to provide energy for the growth of NOB, eventually leading to the elimination of NOB from the bacterial clusters in sequencing batch reactor sludge system., (Copyright © 2015. Published by Elsevier B.V.)

The prerequisite to the development success of the novel mainstream processes partial nitritation/anammox is the out-selection of nitrite oxidizing bacteria (NOB). A recent study suggested that this could be achieved through NO production by ammonium oxidizing bacteria under cyclic oxic-anoxic conditions. Indeed, it is known that among NOB, Nitrobacter species are reversibly inhibited by NO. However, the effect of NO on the activity of the NOB genus Nitrospira is not studied so far. Such an understanding is needed, since Nitrospira related NOB are mostly prevailing in sewage treatment plants. This study quantified the effect of NO on the nitratation activity of sludge types with different Nitrobacter/Nitrospira ratios. In an oxic bubbling column, a dosage of 4.4 mg NO L(-1) d(-1) (∼2 μg NO-N L(-1) in liquid phase) inhibited the Nitrobacter dominated sludge with 24%. For the Nitrospira dominated sludge types, the inhibition was strongly correlated with the nitrite half saturation constant (K(s)) ranging from 0% to 30-50% and 60-80% inhibition of the nitrite oxidation for K(s) of 0.72, 0.36 and 0.06 mg NO2(-)-N L(-1), respectively. This study showed that nitrifying communities with high affinity for nitrite and low specific nitrite oxidation rates (K-strategists) can be strongly inhibited by NO. The degree of inhibition could be confirmed in a set-up with NO dosage through an artificial alginate-based biofilm, ensuring a more direct contact between NO and the microorganisms., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This present study aimed to characterize the bacterial community in a well-established nitrifying reactor by high-throughput sequencing of 16S rRNA amplicons. The laboratory-scale continuous stirred tank reactor has been supplied with ammonium (NH(4)(+)) as sole energy source for over 5 years, while no organic carbon has been added, assembling thus a unique planktonic community with a mean NH(4)(+) removal rate of 86 ± 1.4 mg NH(4)(+)-N/(L d). Results showed a nitrifying community composed of bacteria belonging to Nitrosomonas (relative abundance 11.0%) as the sole ammonia oxidizers (AOB) and Nitrobacter (9.3%) as the sole nitrite oxidizers (NOB). The Alphaproteobacteria (42.3% including Nitrobacter) were the most abundant class within the Proteobacteria (62.8%) followed by the Gammaproteobacteria (9.4%). However, the Betaproteobacteria (excluding AOB) contributed only 0.08%, confirming that Alpha- and Gammaproteobacteria thrived in low-organic-load environments while heterotrophic Betaproteobacteria are not well adapted to these conditions. Bacteroidetes, known to metabolize extracellular polymeric substances produced by nitrifying bacteria and secondary metabolites of the decayed biomass, was the second most abundant phylum (30.8%). It was found that Nitrosomonas and Nitrobacter sustained a broad population of heterotrophs in the reactor dominated by Alpha- and Gammaproteobacteria and Bacteroidetes, in a 1:4 ratio of total nitrifiers to all heterotrophs.

In completely autotrophic nitrogen removal over nitrite (CANON) process, the bioactivity of nitrite-oxidizing bacteria (NOB) should be effectively inhibited. In this study, the stability of four high-rate CANON reactors and the effect of free ammonia (FA) and organic material on NOB community structure were investigated using DGGE. Results suggested that with the increasing of FA, the ratio of total nitrogen removal to nitrate production went up gradually, while the biodiversity of Nitrobacter-like NOB and Nitrospira-like NOB both decreased. When the CANON reactor was transformed to simultaneous partial nitrification, anammox and denitrification (SNAD) reactor by introducing organic material, the denitrifiers and aerobic heterotrophic bacteria would compete nitrite or oxygen with NOB, which then led to the biodiversity decreasing of both Nitrobacter-like NOB and Nitrospira-like NOB. The distribution of Nitrobacter-like NOB and Nitrospira-like NOB were evaluated, and finally effective strategies for suppressing NOB in CANON reactors were proposed., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Nitrobacter winogradskyi Nb-255 is a nitrite-oxidizing bacterium that can grow solely on nitrite (NO2(-)) as a source of energy and nitrogen. In most natural situations, NO2(-) oxidation is coupled closely to ammonium (NH4(+)) oxidation by bacteria and archaea and, conceptually, N. winogradskyi can save energy using NH4(+) to meet its N-biosynthetic requirements. Interestingly, NH4(+) delayed the growth of N. winogradskyi when at concentrations higher than 35 mM, but grew well at concentrations below 25 mM NH4(+) while adjusting the expression of 24% of its genes. Notable genes that changed in expression included those with roles in nitrogen and carbon assimilation. Contrary to expectations, higher expression of glutamate synthase (GOGAT), instead of glutamate dehydrogenase, was detected at higher NH4(+) concentration. Genes in assimilatory NO2(-) metabolism and the degradation of glycogen and biofilm/motility were downregulated when N. winogradskyi was grown in the presence of NH4(+). Nitrobacter winogradskyi grown in medium with 25 mM NH4(+) upregulated genes in post-translational modification, protein turnover, biogenesis and chaperons. The data suggest that N. winogradskyi physiology is modified in the presence of NH4(+) and is likely to be modified during coupled nitrification with NH3 oxidizers., (© FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

The biological treatment of ammonia-rich landfill leachates due to an inadequate C to N ratio requires expensive supplementation of carbon from an external carbon source. In an effort to reduce treatment costs, the objective of the study was to determine the feasibility of nitrogen removal via the nitrite pathway during landfill leachate co-treatment with municipal wastewater. Initially, the laboratory-scale sequencing batch reactor (SBR) was inoculated with nitrifying activated sludge and fed only raw municipal wastewater (RWW) during a start-up period of 9 weeks. Then, in the co-treatment period, consisting of the next 17 weeks, the system was fed a mixture of RWW and an increasing quantity of landfill leachates (from 1 to 10% by volume). The results indicate that landfill leachate addition of up to 10% (by volume) influenced the effluent quality, except for BOD5. During the experiment, a positive correlation (r(2) = 0.908) between ammonia load in the influent and nitrite in the effluent was observed, suggesting that the second step of nitrification was partially inhibited. The partial nitrification (PN) was also confirmed by fluorescence in situ hybridisation (FISH) analysis of nitrifying bacteria. Nitrogen removal via the nitrite pathway was observed when the oxygen concentration ranged from 0.5 to 1.5 mg O2/dm(3) and free ammonia (FA) ranged from 2.01 to 35.86 mg N-NH3/dm(3) in the aerobic phase. Increasing ammonia load in wastewater influent was also correlated with an increasing amount of total nitrogen (TN) in the effluent, which suggested insufficient amounts of assimilable organic carbon to complete denitrification. Because nitrogen removal via the nitrite pathway is beneficial for carbon-limited and highly ammonia-loaded mixtures, obtaining PN can lead to a reduction in the external carbon source needed to support denitrification.

In the activated sludge process, reducing the operational dissolved oxygen (DO) concentration can improve oxygen transfer efficiency, thereby reducing energy use. The low DO, however, may result in incomplete nitrification. This research investigated the long-term effect of low DO on the nitrification performance of activated sludge. Results indicated that, for reactors with 10 and 40 day solids retention times (SRTs), complete nitrification was accomplished after a long-term operation with a DO of 0.37 and 0.16 mg/L, respectively. Under long-term low DO conditions, nitrite oxidizing bacteria (NOB) became a better oxygen competitor than ammonia oxidizing bacteria (AOB) and, as a result, no nitrite accumulated. Real-time PCR assays indicated that the long-term low DO enriched both AOB and NOB in activated sludge, increasing the sludge nitrification capacity and diminishing the adverse effect of low DO on the overall nitrification performance. The increase in the population size of nitrifiers was likely resulted from the reduced nitrifier endogenous decay rate by a low DO. Under long-term low DO conditions, Nitrosomonas europaea/eutropha remained as the dominant AOB, whereas the number of Nitrospira-like NOB became much greater than Nitrobacter-like NOB, especially for the 40 day SRT sludge. The enrichment and shift of the nitrifier community reduced the adverse effect of low DO on nitrification; therefore, low DO operation of a complete nitrification process is feasible.

The dominant nitrifying bacterial communities and nitrification performance of two biological nutrient removal plants were evaluated. Fluorescent in situ hybridization was used to detect and quantify the dominant nitrifying bacteria and polymerase chain reaction; cloning and sequence analysis of 16S rRNA genes was done for phylogenetic analysis. Fluorescent in situ hybridization-confocal scanning laser microscopy studies revealed the presence and dominance of Nitrosomonas-related ammonia-oxidizing bacteria (AOB) and Nitrobacter-related nitrite-oxidizing bacteria (NOB); however, a significant variation in AOB/NOB ratios was recorded. The plant with an overall higher AOB/NOB ratio (> or = 1.0) and dissolved oxygen concentration (1.8 to 2.5 mg/L) showed a higher nitrification rate. This study has also shown the co-existence and variation in phylogenetically diverse Nitrosomonas-related AOB and Nitrobacter-related NOB at these two plants. These dissimilar, distinct distribution patterns of nitrifying communities could be attributed to wastewater characteristics and the process configuration, which, in turn, would have also affected the nitrification performance of the systems.

An up-flow oxygen-controlled biofilm reactor filled with volcanic filter was used for a completely autotrophic nitrogen-removal over nitrite (CANON) process. The reactor was successfully established by seeding conventional activated sludge at ambient temperature without additional biomass inoculation. An average total nitrogen (TN) removal rate of 1.1 kg·(m(3) d)(-1) was achieved after 180 days' operation. The bacterial morphology, community structure and spatial distribution of nitrogen removal microorganisms were analyzed by using some molecular biotechniques. Denaturant gel gradient electrophoresis (DGGE) profiles showed a distinct community shift of nitrite oxidizing bacteria (NOB) during the start-up period, which was not the same as that of aerobic ammonium-oxidizing bacteria (AerAOB) or anaerobic ammonium-oxidizing bacteria (AnAOB). Phylogenetic results indicated the predominance of Nitrosomonas, 'Candidatus Brocadia fulgida' and Nitrobacter for nitrogen removal in the system, all of which coexisted without a distinguishable niche on the biofilm.

The spatial distribution and activities of nitrifying and denitrifying bacteria in sponge media were investigated using diverse tools, because understanding of in situ microbial condition of sponge phase is critical for the successful design and operation of sponge media process. The bacterial consortia within the media was composed of diverse groups including a 14.5% Nitrosomonas spp.-like ammonia oxidizing bacteria (AOB), 12.5% Nitrobacter spp.-like nitrite oxidizing bacteria (NOB), 2.0% anaerobic ammonium-oxidizing (ANAMMOX) bacteria and 71.0% other bacteria. The biofilm appeared to be most dense in the relatively outer region of the media and gradually decreased with depth, but bacterial viabilities showed space-independent feature. The fluorescent in situ hybridization results revealed that AOB and NOB co-existed in similar quantities on the side fragments of the media, which was reasonably supported by the microelectrode measurements showing the concomitant oxidation of NH(4) (+) and production of NO(3) (-) in this zone. However, a significantly higher fraction of AOB was observed in the center than side fragment. As with the overall biofilm density profile, the denitrifying bacteria were also more abundant on the side than in the center fragments. ANAMMOX bacteria detected throughout the entire depth offer another advantage for the removal of nitrogen by simultaneously converting NH(4) (+) and NO(2) (-) to nitrogen gas.

In order to explore the mechanism of NH(4+)-N removal in drinking water biofilter, water quality parameters, such as NH(4+)-N, NO(2-)-N, NO(3-)-N, total phosphorus, permanganate index, nitrogen gas, temperature and dissolved oxygen etc, were determined in the inflow and outflow of biofilter. Samples of granular activated carbon (GAC) at different height (0, 10, 20, 40, 60 cm) of the biofiter media were collected and analyzed for the bacterial community with molecular biology techniques. The bacterial diversity in the activated carbon biofilm sample was studied based on the phylogenetic analysis of sequences. The results showed that there were three stages according to the NH(4+)-N concentration in the influent. The "nitrogen loss" phenomenon (total inorganic nitrogen in the effluent was less than that in the influent) occurred at the first, second and third stages and the amount of nitrogen loss were 0.94, 0.32 and 0.15 mg x L(-1), respectively. The amount of nitrogen loss had a good positive correlation with the NH(4+)-N concentration in the influent, but not a linear relationship with the concentration of the permanganate index in the influent. The average concentrations of N2 increased gradually with the height of media in the biofilter, with values of 14.04 and 14.67 mg x L(-1) in the influent and the effluent, respectively. Based on the sequencing results, the ammonia-oxidizing bacteria (AOB) in the activated carbon biofilm were classified into three common genera: Nitrosococcus, Nitrosomonas and Nitrosospira. When the NH(4+)-N concentration in the influent was relatively high, the "nitrogen loss" phenomenon in biofilter was caused by the AOB.

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.

The crude oil degradability and plasmid profile of autotrophic nitrifying bacteria, Nitrosomonas and Nitrobacter species, isolated from mangrove sediment in the Niger Delta of Nigeria were studied. The effects of temperature, pH and optical density on the utilization of different carbon sources by the bacteria were also investigated. Results showed that nitrifying bacteria could utilize kerosene, diesel oil, jet fuel and engine oil as carbon sources. None utilized hexane and xylene but moderate growth was observed in benzene, phenol and toluene. However, their ability to utilized crude oil varied both in rates of utilization and in growth profiles. Mixed culture of the isolates degrades 52 % of crude oil introduced into the medium followed by Nitrosomonas sp. with 40 % degradation. The least was Nitrobacter sp. with 20 % degradation. The ability of the autotrophs to degrade crude oil was found to be plasmid-mediated through curing experiment and electrophoresis. The size of the plasmid involved was estimated to be 23 kb. The high crude oil utilization of the mixed culture implies that nitrifying bacteria isolated from contaminated ecosystem are excellent crude oil degraders and can be harnessed for bioremediation purposes.

Microorganisms in nitrogen cycle serve as an important part of the ecological function of soil. The aim of this research was to monitor the abundance of nitrogen-fixing, denitrifying and nitrifying bacteria during bioaugmentation of petroleum-contaminated soil using real-time polymerase chain reaction (real-time PCR) of nifH, narG and amoA genes which encode the key enzymes in nitrogen fixation, nitrification and ammoniation respectively. Three different kinds of soils, which are petroleum-contaminated soil, normal soil, and remediated soil, were monitored. It was shown that the amounts of functional microorganisms in petroleum-contaminated soil were far less than those in normal soil, while the amounts in remediated soil and normal soil were comparable. Results of this experiment demonstrate that nitrogen circular functional bacteria are inhibited in petroleum-contaminated soil and can be recovered through bioremediation. Furthermore, copies of the three functional genes as well as total petroleum hydrocarbons (TPH) for soils with six different treatments were monitored. Among all treatments, the one, into which both E. cloacae as an inoculant and wheat straw as an additive were added, obtained the maximum copies of 2.68 x 10(6), 1.71 x 10(6) and 8.54 x 10(4) per gram dry soil for nifH, narG and amoA genes respectively, companying with the highest degradation rate (48% in 40 days) of TPH. The recovery of functional genes and removal of TPH were better in soil inoculated with E cloacae and C echinulata collectively than soil inoculated with E cloacae only. All above results suggest that the nitrogen circular functional genes could be applied to monitor and assess the bioremediation of petroleum-contaminated soil.