Residual Ammonium Concentration Research Articles

One-year stable pilot-scale operation demonstrates high flexibility of mainstream anammox application

Mainstream nitrogen removal via anammox is widely recognized as a promising wastewater treatment process. However, its application is challenging at large scale due to unstable suppression of nitrite-oxidizing bacteria (NOB). In this study, a pilot-scale mainstream anammox process was implemented in an Integrated Fixed-film Activated Sludge (IFAS) configuration. Stable operation with robust NOB suppression was maintained for over one year. This was achieved through integration of three key control strategies: i) low dissolved oxygen (DO = 0.4 ± 0.2 mg O2/L), ii) regular free nitrous acid (FNA)-based sludge treatment, and iii) residual ammonium concentration control (NH4+ with a setpoint of ∼8 mg N/L). Activity tests and FISH demonstrated that NOB barely survived in sludge flocs and were inhibited in biofilms. Despite receiving organic-deficient wastewater from a pilot-scale High-Rate Activated Sludge (HRAS) system as the feed, the system maintained a stable effluent total nitrogen concentration mostly below 10 mg N/L, which was attributed to the successful retention of anammox bacteria. This study successfully demonstrated large-scale long-term mainstream anammox application and generated new practical knowledge for NOB control and anammox retention.

Selective Enrichment of Comammox Nitrospira in a Moving Bed Biofilm Reactor with Sufficient Oxygen Supply.

The recent discovery of comammox (complete ammonia oxidation) Nitrospira has upended the long-held nitrification paradigm. Although comammox Nitrospira have been identified in wastewater treatment systems, the conditions for their dominance over canonical ammonia oxidizers remain unclear. Here, we report the dominance of comammox Nitrospira in a moving bed biofilm reactor (MBBR) fed with synthetic mainstream wastewater. Integrated 16S rRNA gene amplicon sequencing, fluorescence in situ hybridization (FISH), and metagenomic sequencing methods demonstrated the selective enrichment of comammox bacteria when the MBBR was operated at a dissolved oxygen (DO) concentration above 6 mg O2/L. The dominance of comammox Nitrospira over canonical ammonia oxidizers (i.e., Nitrosomonas) was attributed to the low residual ammonium concentration (0.02-0.52 mg N/L) formed in the high-DO MBBR. Two clade A comammox Nitrospira were identified, which are phylogenetically close to Candidatus Nitrospira nitrosa. Interestingly, cryosectioning-FISH showed these two comammox species spatially distributed on the surface of the biofilm. Moreover, the ammonia-oxidizing activity of comammox Nitrospira-dominated biofilms was susceptible to the oxygen supply, which dropped by half with the DO concentration decrease from 6 to 2 mg O2/L. These features collectively suggest a low apparent oxygen affinity for the comammox Nitrospira-dominated biofilms in the high-DO nitrifying MBBR.

Comammox Nitrospira Bacteria Are Dominant Ammonia Oxidizers in Mainstream Nitrification Bioreactors Emended with Sponge Carriers.

Complete ammonia oxidation (i.e., comammox) is a newly discovered microbial process performed by a subset of the Nitrospira genus, and this unique microbial process has been ubiquitously detected in various wastewater treatment units. However, the operational conditions favoring comammox prevalence remain unclear. In this study, the dominance of comammox Nitrospira in four sponge biofilm reactors fed with low-strength ammonium (NH4+ = 23 ± 3 mg N/L) wastewater was proved by coupling 16S rRNA gene amplicon sequencing, quantitative polymerase chain reaction (qPCR), and metagenomic sequencing. The results showed that comammox Nitrospira dominated in the nitrifying guild over canonical ammonia-oxidizing bacteria (AOB) constantly, despite the significant variation in the residual ammonium concentration (0.01-15 mg N/L) under different sets of operating conditions. This result indicates that sponge biofilms greatly favor retaining comammox Nitrospira in wastewater treatment and highlights an essential role of biomass retention in the comammox prevalence. Moreover, analyses of the assembled metagenomic sequences revealed that the retrieved amoA gene sequences affiliated with comammox Nitrospira (53.9-66.0% read counts of total amoA gene reads) were always higher than those (28.4-43.4%) related to β-proteobacterial AOB taxa. The comammox Nitrospira bacteria detected in the present biofilm systems were close to clade A Candidatus Nitrospira nitrosa.

Achieving rapid mainstream deammonification through inoculating long-term refrigerated sidestream sludge in plug-flow fixed-bed biofilm reactor

The start-up of a stable mainstream deammonification requires sufficient anaerobic ammonia-oxidizing bacteria (AnAOB). This study used a plug-flow fixed-bed reactor (PFBR) to verify the feasibility of establishing the mainstream deammonification system by inoculating the sidestream sludge after long-term refrigeration. A rapid resuscitation of the mainstream deammonification system was accomplished by controlling the front-end aeration rate of the PFBR. Results showed that the system was rapidly resuscitated in 44 days eventually with the nitrogen removal rate and nitrogen removal efficiency of 0.1 kg N·(m3·d)−1 and 79.1%, respectively. Also, the efficient performance was secured by the proportionate approaching equilibrium of AnAOB and ammonia-oxidizing bacteria (AOB) activity of 2.35 ± 0.40 and 2.60 ± 0.29 mg N·(g VSS·h)−1, respectively. In addition, Pearson correlation analysis revealed that AnAOB abundance (detected Candidatus Kuenenia) negatively correlated with the AOB (mainly Nitrosomonas)/AnAOB abundance ratio, while correlated positively with the residual ammonium concentration of a region. Furthermore, long-term refrigeration probably reduced the cross-feed relationship between AnAOB and other symbiotic organisms (Armatimonadetes and Chloroflexi) to maintain the basic metabolism. Meanwhile, extracellular polymeric substances produced by other genera (order Xanthomonadales and Pseudomonadales) decreased the mass transfer, protecting AnAOB from unfavorable conditions, thereby facilitating high AnAOB abundance during refrigeration. Thus, this study provides a promising perspective towards the practical applications of mainstream process.

Achieving high-rate partial nitritation with aerobic granular sludge at low temperatures.

Partial nitritation is necessary for the implementation of the mainstream anammox (anaerobic ammonium oxidation) process in wastewater treatment plants. However, the difficulty in outcompeting nitrite-oxidizing bacteria (NOB) at mainstream conditions hinders the performance of partial nitritation. The present work aimed to develop a high-rate partial nitritation process for low-ammonium wastewater treatment at low temperatures by seeding aerobic granules. Experimental results suggested that both stratified structure of nitrifiers developed in the granules and sufficient residual ammonium concentration (18-35mgN L-1) in the bulk liquid contributed to efficient NOB repression. With the hydraulic retention time progressively shortened from 1.0 to 0.17h, the influent nitrogen loading rate of the partial nitritation process reached 6.8 ± 0.4kgN m-3 d-1 even at 10-15°C. The high concentration (7.5 gVSS L-1) and activity (0.48gN g-1 VSS d-1 at 11°C) of granular sludge made the reactor possess an overcapacity evaluated by the ratio between the actual ammonium oxidation rate of the granules and their maximum potential. The overcapacity helped the reactor to face the adverse effect of decreasing temperatures. Overall, this work indicated the great potential of applying aerobic granules to achieve high-rate partial nitritation at mainstream conditions. Moreover, anammox bacteria with a relative abundance of 2.8% was also identified in the partial nitritation granules at the end of this study, suggesting that the granules provided a habitable niche for anammox bacteria growth. Note that these results cannot fully relate to the treatment of real domestic/municipal wastewater, they are a source of important information increasing the knowledge about low temperature partial nitrification.

Achieving stable and efficient single-stage deammonification using plug flow reactor.

The deammonification process is a promising technology, while achieving stable performance is still a challenge for domestic sewage treatment. To investigate the stability of deammonification in the plug flow system, which can be updated from A/O or A/A/O bioreactor, a plug flow fixed biofilm reactor was started-up and fed with synthetic low-strength wastewater. As a result, average ammonium removal efficiency of 90.0 ± 10.0% and total nitrogen removal efficiency of 79.4 ± 9.3% were achieved, while the nitrate production ratio (∆Nitrate/∆Ammonium) was at superior levels (9.5 ± 3.4%). Candidatus Jettenia and Candidatus Brocadia were the anammox bacteria in this reactor, and Candidatus Jettenia was the predominant anammox bacteria. Anammox bacteria were dominated in three of the four sampling points except the first one. Relative abundance of NOB increased along the reactor. The result of the present work implied that the plug flow system was able to maintain stable deammonification process, and NOB was suppressed by higher residual ammonium concentration in the front of reactor while the suppression weakened along the reactor.

Partial Nitrification Fast Start-up and Stable Performance of 15℃ SBBR

In this study, controlled C/N effects on fast start-up and stable performance of partial nitrification process at 15℃ in a Sequenced Batch Biofilm Reactor (SBBR) were investigated. The results showed that partial nitrification successfully fast initiated when C/N was 1.5 but failed when C/N was 0/3 during 60 cycles. Fluorescence in situ hybridization and confocal laser scanning microscope (FISH-CLSM) results showed that ammonia oxidizing bacteria (AOB) was found as the dominant bacteria population when C/N was 1.5. When C/N were 0/3, there were almost no existence of AOB and nitrite oxidative bacteria (NOB). Partial nitrification could be stably achieved without carbon source. However, the addition of an appropriate amount of carbon can effectively improve the nitrification performance, and it is better for the stable operation of partial nitrification. In this experiment, partial nitrification was successfully initiated at high dissolved oxygen (DO) (about 9 mg·L-1) conditions. The average DO was maintained at about 6.5 mg·L-1 during the stable operation, which successfully decoupled partial nitrification from low DO concentration. Excessive residual ammonium concentration in the reactors effectively repressed the growth of NOB and guaranteed the stable operation of partial nitrification. At 15℃, full nitritation was more suitable for sidestream wastewater, while mainstream wastewater was more suitable for partial nitritation.

Effects of the residual ammonium concentration on NOB repression during partial nitritation with granular sludge

Partial nitritation was stably achieved in a bench-scale airlift reactor (1.5L) containing granular sludge. Continuous operation at 20 °C treating low-strength synthetic wastewater (50 mg N-NH4+/L and no COD) achieved nitrogen loading rates of 0.8 g N-NH4+/(L·d) during partial nitritation. The switch between nitrite-oxidizing bacteria (NOB) repression and NOB proliferation was observed when ammonium concentrations in the reactor were below 2–5 mg N-NH4+/L for DO concentrations lower than 4 mg O2/L at 20 °C. Nitrospira spp. were detected to be the dominant NOB population during the entire reactor operation, whereas Nitrobacter spp. were found to be increasing in numbers over time. Stratification of the granule structure, with ammonia-oxidizing bacteria (AOB) occupying the outer shell, was found to be highly important in the repression of NOB in the long term. The pH gradient in the granule, containing a pH difference of ca. 0.4 between the granule surface and the granule centre, creates a decreasing gradient of ammonia towards the centre of the granule. Higher residual ammonium concentration enhances the ammonium oxidation rate of those cells located further away from the granule surface, where the competition for oxygen between AOB and NOB is more important, and it contributes to the stratification of both populations in the biofilm.

Analysing the effects of the aeration pattern and residual ammonium concentration in a partial nitritation-anammox process

ABSTRACTA mathematical model was used to evaluate the effect of the aeration pattern and ammonium concentration in a partial nitritation-anammox sequencing batch reactor with granular and flocculent sludge. In the tested conditions, model results indicate that most of the aerobic ammonium oxidation potential would occur in the bulk liquid, with 70% of the ammonium-oxidizing bacteria (AOB) biomass in suspension rather than in granules. The simulated granular sludge consisted predominantly of anammox bacteria with AOB present in the outer layer of the granule (50 μm AOB layer, accounting for 3% of the granule weight). Simulation results indicated that when granules do not contain any AOB, the amount of granular biomass required to achieve the same level of nitrogen removal would strongly increase (in the simulated conditions, by a factor of three) due to anammox inhibition by oxygen. This underlines the importance of a small fraction of AOB present in the granular anammox sludge. The aeration pattern had an important impact on the nitrogen removal: a better performance was suggested for continuous aeration (90% N-removal) than for intermittent aeration (68–84% N-removal). Anammox inhibition during the periods of high oxygen concentration was identified as the main reason for the lower nitrogen removal in the intermittently aerated system. With increasing oxygen concentration, a higher residual (effluent) ammonium concentration was needed to assure nitrite-oxidizing bacteria repression in the system. This study contributes to further understand the complexity of a reactor with both granular and flocculent sludge and the impact of operation conditions on reactor performance.

Outcompeting nitrite-oxidizing bacteria in single-stage nitrogen removal in sewage treatment plants: A model-based study

This model-based study investigated the mechanisms and operational window for efficient repression of nitrite oxidizing bacteria (NOB) in an autotrophic nitrogen removal process. The operation of a continuous single-stage granular sludge process was simulated for nitrogen removal from pretreated sewage at 10 °C. The effects of the residual ammonium concentration were explicitly analyzed with the model. Competition for oxygen between ammonia-oxidizing bacteria (AOB) and NOB was found to be essential for NOB repression even when the suppression of nitrite oxidation is assisted by nitrite reduction by anammox (AMX). The nitrite half-saturation coefficient of NOB and AMX proved non-sensitive for the model output. The maximum specific growth rate of AMX bacteria proved a sensitive process parameter, because higher rates would provide a competitive advantage for AMX.

Mimicking the oxygen minimum zones: stimulating interaction of aerobic archaeal and anaerobic bacterial ammonia oxidizers in a laboratory‐scale model system

In marine oxygen minimum zones (OMZs), ammonia-oxidizing archaea (AOA) rather than marine ammonia-oxidizing bacteria (AOB) may provide nitrite to anaerobic ammonium-oxidizing (anammox) bacteria. Here we demonstrate the cooperation between marine anammox bacteria and nitrifiers in a laboratory-scale model system under oxygen limitation. A bioreactor containing ‘Candidatus Scalindua profunda’ marine anammox bacteria was supplemented with AOA (Nitrosopumilus maritimus strain SCM1) cells and limited amounts of oxygen. In this way a stable mixed culture of AOA, and anammox bacteria was established within 200 days while also a substantial amount of endogenous AOB were enriched. ‘Ca. Scalindua profunda’ and putative AOB and AOA morphologies were visualized by transmission electron microscopy and a C18 anammox [3]-ladderane fatty acid was highly abundant in the oxygen-limited culture. The rapid oxygen consumption by AOA and AOB ensured that anammox activity was not affected. High expression of AOA, AOB and anammox genes encoding for ammonium transport proteins was observed, likely caused by the increased competition for ammonium. The competition between AOA and AOB was found to be strongly related to the residual ammonium concentration based on amoA gene copy numbers. The abundance of archaeal amoA copy numbers increased markedly when the ammonium concentration was below 30 μM finally resulting in almost equal abundance of AOA and AOB amoA copy numbers. Massive parallel sequencing of mRNA and activity analyses further corroborated equal abundance of AOA and AOB. PTIO addition, inhibiting AOA activity, was employed to determine the relative contribution of AOB versus AOA to ammonium oxidation. The present study provides the first direct evidence for cooperation of archaeal ammonia oxidation with anammox bacteria by provision of nitrite and consumption of oxygen.

Precipitation of Ammonium from Concentrated Industrial Wastes as Struvite: A Search for the Optimal Reagents

Precipitation of struvite (MgNH4PO4) is a known process for purification of wastewater from high concentrations of ammonium. The optimal conditions for precipitation are basic pH (around 9) and sufficient concentrations of magnesium and phosphate ions. In this work, we accomplished efficient precipitation of ammonium from concentrated industrial waste stream by using magnesium oxide (MgO) both as a source of magnesium ions and as a base. Best results were obtained with technical-grade MgO, which provided 99% removal of ammonium. Moreover, ammonium removal occurred already at pH 7, and the residual ammonium concentration (50 mg/L) remained constant upon addition of more MgO without rising again, as occurs with sodium hydroxide (NaOH). This process may have two other advantages; it also can be relevant for the problem of uncontrolled precipitation of struvite in the supernatant of anaerobic sludge treatment plants, and the precipitate can be used as a fertilizer.

Ammonium removal from aqueous solutions by dissolved air flotation in the presence of zeolite carrier

In this paper, dissolved air flotation (DAF) was examined as a possible treatment method for the removal of ammonium ions (NH 4 +) from surface water. A natural zeolite, clinoptilolite sample with a fine particle size (< 10 µm) was used as a carrier material during the flotation tests. Two types DAF cells, batch and continuous implemented in our laboratories, were used in the flotation experiments. Flotation tests revealed that the test loaded with 5 mg/L ammonium resulted in a residual ammonium concentration of < 0.5 mg/L with corresponding turbidity values of treated water (< 10 NTU). It was observed that the usage of carrier material with higher treatment capacity and finer ion exchange material of higher surface area than classical ion exchangers may provide better advantages over the classical DAF method.

Production of Medium-Chain-Length Poly(3-hydroxyalkanoates) from Crude Fatty Acids Mixture by Pseudomonas putida

Microbial production of medium-chain-length poly(3-hydroxyalkanoates), PHA MCL from crude fatty acids mixture was investigated. With ammonium as the growth limiting substrate, fatty acids mixture from saponified palm kernel oil (SPKO) supports good growth and PHA MCL production of Pseudomonas putida PGA1. Growth of this microorganism on ammonium exhibited substrate inhibition kinetics which can be described using Andrews model with the substrate inhibition constant, K i = 1.2 g L −1. Concentration of SPKO in the aqueous medium should be at 10 g L −1 or less, as higher concentrations can significantly reduced the volumetric oxygen transfer coefficient (K L a), biomass growth and PHA MCL production. Uptake of SPKO by the organism follows a zero-order kinetics, indicating a mass transfer limitation of the free fatty acids by the P. putida PGA1 cells. In batch and fed-batch fermentations, PHA MCL accumulation is encouraged under ammonium-limited condition with SPKO as the sole carbon and energy source. The amount of PHA MCL accumulated and its specific production rate, q PHA were influenced by the residual ammonium concentration level in the culture medium. It was observed that in both fermentation modes, when the residual ammonium becomes exhausted (<0.05 g L −1), the PHA MCL accumulation and q PHA were significantly reduced. However, this effect can be reversed by feeding low amount of ammonium to the culture, resulting in significantly improved PHA MCL yield and productivity. It is concluded that the feeding of residual ammonium concentration in the culture medium during the PHA MCL accumulation has a positive effect on sustaining the PHA MCL biosynthetic capability of the organism.

Production of Medium-Chain-Length Poly(3-Hydroxyalkanoates) from Saponified Palm Kernel Oil by Pseudomonas putida: Kinetics of Batch and Fed-Batch Fermentations

The kinetics of medium-chain-length poly(3-hydroxyalkanoates), PHAMCL production by Pseudomonas putida PGA1 in batch and fed-batch fermentations were studied. With saponified palm kernel oil (SPKO) supplying the free fatty acids mixture as the sole carbon and energy source, PHAMCL accumulation is encouraged under ammonium-limited condition, which is a nitrogen stress environment. The amount of PHAMCL accumulated and its specific production rate, qPHA were influenced by the residual ammonium concentration level in the culture medium. It was observed that in both fermentation modes, when the residual ammonium was exhausted (< 0.05 gL-1), the PHAMCL accumulation (11.9) and qPHA (0.0062 h-1) were significantly reduced. However, this effect can be reversed by feeding low amount of ammonium to the culture, resulting in significantly improved PHAMCL yield (71.4) and specific productivity (0.6 h-1). It is concluded that the feeding of low ammonium concentration to the culture medium during the PHAMCL accumulation has a positive effect on sustaining the PHAMCL biosynthetic capability of the organism. It was also found that increasing SPKO concentration in the medium significantly reduced (up to 50) the volumetric oxygen transfer coefficient (KLa) of the fermentation system.

Ammonium Limitation Results in the Loss of Ammonia-Oxidizing Activity in Nitrosomonas europaea.

The effects of limiting concentrations of ammonium on the metabolic activity of Nitrosomonas europaea, an obligate ammonia-oxidizing soil bacterium, were investigated. Cells were harvested during late logarithmic growth and were incubated for 24 h in growth medium containing 0, 15, or 50 mM ammonium. The changes in nitrite production and the rates of ammonia- and hydroxylamine-dependent oxygen consumption were monitored. In incubations without ammonium, there was little change in the ammonia oxidation activity after 24 h. With 15 mM ammonium, an amount that was completely consumed, there was an 85% loss of the ammonia oxidation activity after 24 h. In contrast, there was only a 35% loss of the ammonia oxidation activity after 24 h in the presence of 50 mM ammonium, an amount that was not consumed to completion. There was little effect on the hydroxylamine oxidation activity in any of the incubations. The loss of ammonia oxidation activity was not due to differences in steady-state levels of ammonia monooxygenase (AMO) mRNA (amoA) or to degradation of the active site-containing subunit of AMO protein. The incubations were also conducted at a range of pH values to determine whether the loss of ammonia oxidation activity was correlated to the residual ammonium concentration. The loss of ammonia oxidation activity after 24 h was less at lower pH values (where the unoxidized ammonium concentration was higher). When added in conjunction with limiting ammonium, short-chain alkanes, which are alternative substrates for AMO, prevented the loss of ammonia oxidation activity at levels corresponding to their binding affinity for AMO. These results suggest that substrates of AMO can preserve the ammonia-oxidizing activity of N. europaea in batch incubations by protecting either AMO itself or other molecules associated with ammonia oxidation.

Design of a nitrifying tertiary trickling filter based on theoretical concepts

A nitrification model for a tertiary trickling filter is developed based on stoichiometry, Fick's Law and Monod kinetics. The design of tertiary trickling filters for nitrification is discussed, with special emphasis on: residual ammonium concentration, recirculation, reactors in series, residual alkalinity, residual nitrite concentration and effects of temperature on reactor performance. Wherever possible the theoretical predictions are compared with experimental results.

Regulation of Asparaginase, Glutamine Synthetase, and Glutamate Dehydrogenase in Response to Medium Nitrogen Concentrations in a Euryhaline Chlamydomonas Species

The ammonium assimilatory enzymes glutamine synthetase (EC 6.3.1.2) and glutamate dehydrogenase (EC 1.4.1.3) were investigated for a possible role in the regulation of asparaginase (EC 3.5.1.1) in a Chlamydomonas species isolated from a marine environment. Cells grown under nitrogen limitation (0.1 millimolar NH(4) (+), NO(3) (-), or l-asparagine) possessed 6 times the asparaginase activity and approximately one-half the protein of cells grown at high nitrogen levels (1.5 to 2.5 millimolar). Biosynthetic glutamine synthetase activity was 1.5 to 1.8 times greater in nitrogen-limited cells than cells grown at high levels of the three nitrogen sources.Conversely, glutamate dehydrogenase (both NADH- and NADPH-dependent activities) was greatest in cells grown at high levels of asparagine or ammonium, while nitrate-grown cells possessed little activity at all concentrations employed. For all three nitrogen sources, glutamate dehydrogenase activity was correlated to the residual ammonium concentration of the media after growth (r = 0.88 and 0.94 for NADH- and NADPH-dependent activities, respectively).These results suggest that glutamate dehydrogenase is regulated in response to ambient ammonium levels via a mechanism distinct from asparaginase or glutamine synthetase. Glutamine synthetase and asparaginase, apparently repressed by high levels of all three nitrogen sources, are perhaps regulated by a common mechanism responding to intracellular nitrogen depletion, as evidenced by low cellular protein content.