Growth of the Nitrosomonas europaea cells in the biofilm and planktonic growth mode: Responses of extracellular polymeric substances production and transcriptome.

Nitrosomonas europaea, an aerobic ammonia oxidizing bacterium, is responsible for the first and rate-limiting step of the nitrification process, and their ammonia oxidation activities are critical for the biogeochemical cycling and the biological nitrogen removal of wastewater treatment. In the present study, N. europaea cells were cultivated in the inorganic or organic media (the NBRC829 and the nutrient-rich, NR, media, respectively), and the cells proliferated in the form of planktonic and biofilm in those media, respectively. The N. europaea cells in the biofilm growth mode produced larger amounts of the extracellular polymeric substances (EPS), and the composition of the EPS was characterized by the chemical analyses including Fourier transform infrared spectroscopy (FT-IR) and 1H-nuclear magnetic resonance (NMR) measurements. The RNA-Seq analysis of N. europaea in the biofilm or planktonic growth mode revealed that the following gene transcripts involved in central nitrogen metabolisms were abundant in the biofilm growth mode; amo encoding ammonia monooxygenase, hao encoding hydroxylamine dehydrogenase, the gene encoding nitrosocyanine, nirK encoding copper-containing nitrite reductase. Additionally, the transcripts of the pepA and wza involved in the bacterial floc formation and the translocation of EPS, respectively, were also abundant in the biofilm-growth mode. Our study was first to characterize the EPS production and transcriptome of N. europaea in the biofilm and planktonic growth mode.

Flux balance analysis of the ammonia-oxidizing bacterium Nitrosomonas europaea ATCC19718 unravels specific metabolic activities while degrading toxic compounds.

The ammonia-oxidizing bacterium Nitrosomonas europaea has been widely recognized as an important player in the nitrogen cycle as well as one of the most abundant members in microbial communities for the treatment of industrial or sewage wastewater. Its natural metabolic versatility and extraordinary ability to degrade environmental pollutants (e.g., aromatic hydrocarbons such as benzene and toluene) enable it to thrive under various harsh environmental conditions. Constraint-based metabolic models constructed from genome sequences enable quantitative insight into the central and specialized metabolism within a target organism. These genome-scale models have been utilized to understand, optimize, and design new strategies for improved bioprocesses. Reduced modeling approaches have been used to elucidate Nitrosomonas europaea metabolism at a pathway level. However, genome-scale knowledge about the simultaneous oxidation of ammonia and pollutant metabolism of N. europaea remains limited. Here, we describe the reconstruction, manual curation, and validation of the genome-scale metabolic model for N. europaea, iGC535. This reconstruction is the most accurate metabolic model for a nitrifying organism to date, reaching an average prediction accuracy of over 90% under several growth conditions. The manually curated model can predict phenotypes under chemolithotrophic and chemolithoorganotrophic conditions while oxidating methane and wastewater pollutants. Calculated flux distributions under different trophic conditions show that several key pathways are affected by the type of carbon source available, including central carbon metabolism and energy production.

A two-lane mechanism for selective biological ammonium transport.

The transport of charged molecules across biological membranes faces the dual problem of accommodating charges in a highly hydrophobic environment while maintaining selective substrate translocation. This has been the subject of a particular controversy for the exchange of ammonium across cellular membranes, an essential process in all domains of life. Ammonium transport is mediated by the ubiquitous Amt/Mep/Rh transporters that includes the human Rhesus factors. Here, using a combination of electrophysiology, yeast functional complementation and extended molecular dynamics simulations, we reveal a unique two-lane pathway for electrogenic NH4+ transport in two archetypal members of the family, the transporters AmtB from Escherichia coli and Rh50 from Nitrosomonas europaea. The pathway underpins a mechanism by which charged H+ and neutral NH3 are carried separately across the membrane after NH4+ deprotonation. This mechanism defines a new principle of achieving transport selectivity against competing ions in a biological transport process.

Impacts of anthropogenic gadolinium on the activity of the ammonia oxidizing bacterium Nitrosomonas europaea.

Widespread use of gadolinium-based contrast agents in medical imaging has resulted in increased Gd inputs to municipal wastewater treatment plants. Others have reported that typical wastewater treatment does not attenuate Gd, resulting in discharges to natural waters. However, whether elevated Gd impacts the performance of biological treatment has not been investigated. We examined whether gadolinium chloride or Gd chelated with diethylenetriaminepentaacetic acid (DTPA) affected the activity of the model nitrifying bacterium Nitrosomonas europaea. At nominal GdCl3 additions ranging from 1 to 500 µM, no impact was observed compared to the control. Most (>98%) of the added Gd precipitated, and extracellular GdPO4 nanoparticles were observed. When chelated with DTPA, Gd remained soluble, but no statistically significant impact on ammonia oxidation was observed until the highest concentrations tested. At 300 and 500 µM Gd-DTPA, a temporary reduction of nitrite production relative to the control (effect size 1.3 mg l-1 and 1.5 mg l-1, respectively, at 24 h) was seen. By itself, DTPA was highly inhibitory. Modeling suggested that DTPA likely chelated other metals, but adjusting the concentrations of the most abundant metals in the medium, calcium and magnesium, indicated that lowering their free ion activities was probably not the cause of inhibition. Complexation of other essential metals was more likely. Our studies indicate that while the low bioavailability of Gd may limit its ecosystem impacts, the role of synthetic ligands used with Gd and other rare earth elements should be considered as the production, use and disposal of these elements increases.

Engineered small metal-binding protein tag improves the production of recombinant human growth hormone in the periplasm of Escherichia coli.

Fusion proteins play an important role in the production of recombinant proteins in Escherichia coli. They are mostly used for cytoplasmic expression since they can be designed to increase the solubility of the target protein, which then can be easily purified via affinity chromatography. In contrast, fusion proteins are not usually included in construct designs for periplasmic production. Instead, a signal sequence is inserted for protein transport into the periplasm and a C-terminal his-tag added for subsequent purification. Our research group has proposed the small metal-binding protein (SmbP) isolated from the periplasm of Nitrosomonas europaea as a new fusion protein to express recombinant proteins in the cytoplasm or periplasm of E. coli. SmbP also allows purification via immobilized metal affinity chromatography using Ni(II) ions. Recently, we have optimized the periplasmic production of proteins tagged with SmbP by exchanging its native signal peptide with one taken from pectate lyase B (PelB), substantially increasing the amount of protein produced. In this work, we have expressed and purified soluble bioactive human growth hormone (hGH) tagged with PelB-SmbP and obtained the highest periplasmic production reported for this protein so far. Its activity, tested on Nb2-11 cells, was equivalent to commercial growth hormone at 50 ng·mL-1 . Therefore, we strongly recommend the use of PelB-SmbP as a protein tag for the expression and purification of hGH or other possible target proteins in the periplasm of E. coli.

Detection and Quantification of Nitrifying Bacteria Using Real-Time PCR.

Nitrification is the microbial-mediated transformation of ammonium (NH4+) into nitrate (NO3-). Many plant species depend on the availability of NO3- as the main source of nitrogen (N). On the other hand, because NO3- is highly mobile in the soil profile, its excess concentration can cause environmental pollution. Nitrification can be estimated at the process level, but with the development of molecular techniques it is also possible to estimate the abundance of nitrifying bacteria in the soil. Hence, in this chapter we describe the procedure for detection and quantification of nitrifying bacteria in soil samples using real-time quantitative polymerase chain reaction (PCR).

Urine nitrification with a synthetic microbial community.

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

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

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

Effects of Exogenous N-Acyl-Homoserine Lactone as Signal Molecule on Nitrosomonas Europaea under ZnO Nanoparticle Stress.

Despite the adverse effects of emerging ZnO nanoparticles (nano-ZnO) on wastewater biological nitrogen removal (BNR) systems being widely documented, strategies for mitigating nanoparticle (NP) toxicity impacts on nitrogen removal have not been adequately addressed. Herein, N-acyl-homoserine lactone (AHL)-based quorum sensing (QS) was investigated for its effects against nano-ZnO toxicity to a model nitrifier, Nitrosomonas europaea. The results indicated that AHL-attenuated nano-ZnO toxicity, which was inversely correlated with the increasing dosage of AHL from 0.01 to 1 µM. At 0.01 µM, AHL notably enhanced the tolerance of N. europaea cells to nano-ZnO stress, and the inhibited cell proliferation, membrane integrity, ammonia oxidation rate, ammonia monooxygenase activity and amoA gene expression significantly increased by 18.2 ± 2.1, 2.4 ± 0.9, 58.7 ± 7.1, 32.3 ± 1.7, and 7.3 ± 5.9%, respectively, after 6 h of incubation. However, increasing the AHL dosage compromised the QS-mediated effects and even aggravated the NPs' toxicity effects. Moreover, AHLs, at all tested concentrations, significantly increased superoxide dismutase activity, indicating the potential of QS regulations to enhance cellular anti-oxidative stress capacities when facing NP invasion. These results provide novel insights into the development of QS regulation strategies to reduce the impact of nanotoxicity on BNR systems.

Optimizing Periplasmic Expression in Escherichia coli for the Production of Recombinant Proteins Tagged with the Small Metal-Binding Protein SmbP.

We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.

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

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

Nitrosomonas europaea adaptation to anoxic-oxic cycling: Insights from transcription analysis, proteomics and metabolic network modeling.

In suboxic or anoxic environments, nitrous oxide (N2O) can be produced by ammonia oxidizing bacteria (AOB) as a potent greenhouse gas. Although N2O producing inventory and pathways have been well-characterized using archetypal AOB, there is little known about their adaptive responses to oxic-anoxic cycling, which is a prevalent condition in soil, sediment, and wastewater treatment bioreactors. In this study, cellular responses of Nitrosomonas europaea 19718 to sustained anoxic-oxic cycling in a chemostat bioreactor were evaluated at transcriptomic, proteomic, and fluxomic levels. During a single oxic-anoxic transition, the accumulations of major intermediates were found at the beginning of anoxia (nitric oxide, NO) and post anoxia (hydroxylamine, NH2OH, and N2O). Anoxic-oxic cycling over thirteen days led to significantly reduced accumulations of NH2OH, NO and N2O. Distinct from short-term responses, which were mostly regulated at the mRNA level, adapted cells seemed to sustain energy generation under repeated anoxia by partially sacrificing the NO detoxification capacities, and such adaptation was mainly regulated at the protein level. The proteomic data also suggested the potential contributions of the newly discovered cytochrome P460-mediated NH2OH oxidation pathway to N2O productions. Flux balance analysis was performed based on a metabolic network model consisting of 49 biochemical reactions involved in nitrogen respiration, and changes in metabolic fluxes after the anoxic-oxic cycling were found to be better correlated with intracellular protein concentrations rather than mRNA levels. Previous studies focusing on single anoxic-oxic transition might have overlooked the adaptive responses of nitrifiers to anoxic-oxic cycling, and thus overestimated NO and N2O emission levels from natural and engineered nitrification systems.

Effects of the chemical characteristics and concentration of inorganic suspended solids on nitrification in freshwater.

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 (kNH4) was significantly affected by the ISS type, whereas changes in the ISS concentration had an insignificant effect on kNH4, except for kaolinite. The highest kNH4 was observed in samples containing sericite (kNH4, 0.067 L mg-1 day-1), followed by samples containing montmorillonite (kNH4, 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 (kNO2, 0.13-0.21 L mg-1 day-1) were not significantly different among the three types of ISS, but kNO2 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.

Kinetics of NH3 -oxidation, NO-turnover, N2 O-production and electron flow during oxygen depletion in model bacterial and archaeal ammonia oxidisers.

Ammonia oxidising bacteria (AOB) are thought to emit more nitrous oxide (N2 O) than ammonia oxidising archaea (AOA), due to their higher N2 O yield under oxic conditions and denitrification in response to oxygen (O2 ) limitation. We determined the kinetics of growth and turnover of nitric oxide (NO) and N2 O at low cell densities of Nitrosomonas europaea (AOB) and Nitrosopumilus maritimus (AOA) during gradual depletion of TAN (NH3 + NH4+) and O2 . Half-saturation constants for O2 and TAN were similar to those determined by others, except for the half-saturation constant for ammonium in N. maritimus (0.2 mM), which is orders of magnitudes higher than previously reported. For both strains, cell-specific rates of NO turnover and N2 O production reached maxima near O2 half-saturation constant concentration (2-10 µM O2 ) and decreased to zero in response to complete O2 -depletion. Modelling of the electron flow in N. europaea demonstrated low electron flow to denitrification (≤1.2% of the total electron flow), even at sub-micromolar O2 concentrations. The results corroborate current understanding of the role of NO in the metabolism of AOA and suggest that denitrification is inconsequential for the energy metabolism of AOB, but possibly important as a route for dissipation of electrons at high ammonium concentration.

Nitrifier-induced denitrification is an important source of soil nitrous oxide and can be inhibited by a nitrification inhibitor 3,4-dimethylpyrazole phosphate.

Soil ecosystem represents the largest contributor to global nitrous oxide (N2 O) production, which is regulated by a wide variety of microbial communities in multiple biological pathways. A mechanistic understanding of these N2 O production biological pathways in complex soil environment is essential for improving model performance and developing innovative mitigation strategies. Here, combined approaches of the 15 N-18 O labelling technique, transcriptome analysis, and Illumina MiSeq sequencing were used to identify the relative contributions of four N2 O pathways including nitrification, nitrifier-induced denitrification (nitrifier denitrification and nitrification-coupled denitrification) and heterotrophic denitrification in six soils (alkaline vs. acid soils). In alkaline soils, nitrification and nitrifier-induced denitrification were the dominant pathways of N2 O production, and application of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) significantly reduced the N2 O production from these pathways; this is probably due to the observed reduction in the expression of the amoA gene in ammonia-oxidizing bacteria (AOB) in the DMPP-amended treatments. In acid soils, however, heterotrophic denitrification was the main source for N2 O production, and was not impacted by the application of DMPP. Our results provide robust evidence that the nitrification inhibitor DMPP can inhibit the N2 O production from nitrifier-induced denitrification, a potential significant source of N2 O production in agricultural soils.

Nitric oxide is an obligate bacterial nitrification intermediate produced by hydroxylamine oxidoreductase.

Ammonia (NH3)-oxidizing bacteria (AOB) emit substantial amounts of nitric oxide (NO) and nitrous oxide (N2O), both of which contribute to the harmful environmental side effects of large-scale agriculture. The currently accepted model for AOB metabolism involves NH3 oxidation to nitrite (NO2-) via a single obligate intermediate, hydroxylamine (NH2OH). Within this model, the multiheme enzyme hydroxylamine oxidoreductase (HAO) catalyzes the four-electron oxidation of NH2OH to NO2- We provide evidence that HAO oxidizes NH2OH by only three electrons to NO under both anaerobic and aerobic conditions. NO2- observed in HAO activity assays is a nonenzymatic product resulting from the oxidation of NO by O2 under aerobic conditions. Our present study implies that aerobic NH3 oxidation by AOB occurs via two obligate intermediates, NH2OH and NO, necessitating a mediator of the third enzymatic step.

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

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

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

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

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

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