Nitritation-anammox Process Research Articles

Microbial community structure and biodiversity of size-fractionated granules in a partial nitritation-anammox process.

The performance of a granule-based partial nitritation-anammox process is expected to be affected by the granule size distribution, but little is known about the impact of granule size on microbial community structure and diversity. To reveal how the microbial composition and diversity vary with granule size, granules from a partial nitritation-anammox reactor were size-fractionated into five classes (<0.2, 0.2-0.5, 0.5-0.8, 0.8-1.0 and >1.0 mm). Microbial communities and diversity in these size-fractionated granules were investigated using 16S rRNA gene high-throughput sequencing. It was found that larger granules harbor more diverse microbial communities than small granules. Both quantitative PCR and 16S rRNA gene sequencing indicated that the abundance of anammox bacteria (dominated by Candidatus Brocadia) exhibited an increasing trend with granule size. In contrast, the abundance of ammonia-oxidizing bacteria (Nitrosomonas) decreased with increasing granule size. Moreover, larger granules harbored more diverse anammox bacteria, with four genera found in the largest granules while only two with limited abundance were detected in the smallest granules. The findings highlight an important role for granule size in shaping community structure and biodiversity.

Biomass aggregation influences NaN3 short-term effects on anammox bacteria activity.

The main bottleneck to maintain the long-term stability of the partial nitritation-anammox processes, especially those operated at low temperatures and nitrogen concentrations, is the undesirable development of nitrite oxidizing bacteria (NOB). When this occurs, the punctual addition of compounds with the capacity to specifically inhibit NOB without affecting the process efficiency might be of interest. Sodium azide (NaN3) is an already known NOB inhibitor which at low concentrations does not significantly affect the ammonia oxidizing bacteria (AOB) activity. However, studies about its influence on anammox bacteria are unavailable. For this reason, the objective of the present study was to evaluate the effect of NaN3 on the anammox activity. Three different types of anammox biomass were used: granular biomass comprising AOB and anammox bacteria (G1), anammox enriched granules (G2) and previous anammox granules disaggregated (F1). No inhibitory effect of NaN3 was measured on G1 sludge. However, the anammox activity decreased in the case of G2 and F1. Granular biomass activity was less affected (IC50 90 mg/L, G2) than flocculent one (IC50 5 mg/L, F1). Summing up, not only does the granular structure protect the anammox bacteria from the NaN3 inhibitory effect, but also the AOB act as a barrier decreasing the inhibition.

In-situ restoration of one-stage partial nitritation-anammox process deteriorated by nitrate build-up via elevated substrate levels.

The one-stage partial nitritation and anammox process (PN/A) has been a promising microbial process to remove ammonia from wastewater especially with low carbon/nitrogen ratio. The main breakdown was the deterioration caused by overgrowth of nitrite oxidizing bacteria (NOB) resulting effluent nitrate build-up in the PN/A process. This study presented an in-situ restoring strategy for suppressing NOB activity in a one-stage granular PN/A system deteriorated over 2 months, using elevated concentrations of substrates (ammonia and nitrite) under limited dissolved oxygen level. The results showed that the NOB activity was successfully suppressed after 56 days of restoration, and finally the ratio of produced nitrate/consumed ammonium was reduced from 36.8% to 7%. On day 66 the nitrogen removal rate obtained as 1.2 kg N/(m3·d). The high FA level (5–40 mg/L) and low dissolved oxygen (<0.13 mg/L) were responsible for NOB suppression. From quantitative PCR (qPCR) analysis, after this restoration, anammox bacteria had a widely growth, and AOB stay stable, but Nitrospira increase and Nitrobacter declined. High amount of NOB was still persistent in the granules, which was not easy to wash-out and threaten the deammonification performance.

Fate of 17β-Estradiol as a model estrogen in source separated urine during integrated chemical P recovery and treatment using partial nitritation-anammox process

Recently, research on source separation followed by the treatment of urine and/or resource recovery from human urine has shown promise as an emerging management strategy. Despite contributing only 1% of the total volume of wastewater, human urine contributes about 80% of the nitrogen, 70% of the potassium, and up to 50% of the total phosphorus in wastewater. It is also a known fact that many of the micropollutants, especially selected estrogens, get into municipal wastewater through urine excretion. In this research, we investigated the fate of 17β-estradiol (E2) as a model estrogen during struvite precipitation from synthetic urine followed by the treatment of urine using a partial nitritation-anammox (PN/A) system. Single-stage and two-stage suspended growth PN/A configurations were used to remove the nitrogen in urine after struvite precipitation. The results showed an almost 95% phosphorous and 5% nitrogen recovery/removal from the synthetic urine due to struvite precipitation. The single and two stage PN/A processes were able to remove around 50% and 75% of ammonia and nitrogen present in the post struvite urine solution, respectively. After struvite precipitation, more than 95% of the E2 remained in solution and the transformation of E2 to E1 happened during urine storage. Most of the E2 removal that occurred during the PN/A process was due to sorption on the biomass and biodegradation (transformation of E2 to E1, and slow degradation of E1 to other metabolites). These results demonstrate that a combination of chemical and biological unit processes will be needed to recover and manage nutrients in source separated urine.

Step-wise temperature decreasing cultivates a biofilm with high nitrogen removal rates at 9°C in short-term anammox biofilm tests

ABSTRACTThe anaerobic ammonium oxidation (anammox) and nitritation-anammox (deammonification) processes are widely used for N-rich wastewater treatment. When deammonification applications move towards low temperature applications (mainstream wastewater has low temperature), temperature effect has to be studied. In current research, in a deammonification moving bed biofilm reactor a maximum total nitrogen removal rate (TNRR) of 1.5 g N m−2 d−1 (0.6 kg N m−3 d−1) was achieved. Temperature was gradually lowered by 0.5°C per week, and a similar TNRR was sustained at 15°C during biofilm cultivation. Statistical analysis confirmed that a temperature decrease from 20°C down to 15° did not cause instabilities. Instead, TNRR rose and treatment efficiency remained stable at lower temperatures as well. Quantitative polymerase chain reaction analyses showed an increase in Candidatus Brocadia quantities from 5 × 103 to 1 × 107 anammox gene copies g−1 total suspended solids (TSS) despite temperature lowered to 15°C. Fluctuations in TNRR were rather related to changes in influent concentration. To study the short-term effect of temperature on the TNRR, a series of batch-scale experiments were performed which showed sufficient TNRRs even at 9–15°C (1.24–3.43 mg N g−1 TSS h−1, respectively) with anammox temperature constants (Q10) ranging 1.3–1.6. Experiments showed that a biofilm adapted to 15°C can perform N-removal most sufficiently at temperatures down to 9°C as compared with biofilm adapted to higher temperature. After biomass was adapted to 15°C, the decrease in TNRR in batch tests at 9°C was lower (15–20%) than that for biomass adapted to 17–18°C.

Nitrogen removal by granular nitritation–anammox in an upflow membrane-aerated biofilm reactor

The nitritation–anammox process has been a promising nitrogen removal technology towards sustainable wastewater treatment, but its application in treating domestic wastewater with relatively low ammonium concentrations (mainstream) remains a great challenge. In this study, an innovative lab-scale upflow membrane-aerated biofilm reactor (UMABR) was employed to treat a synthetic wastewater containing 70 mg N L−1 ammonium. With a DO level at 0.6 ± 0.1 mg O2 L−1 and HRT of 32 h, the effluent ammonium concentration was 4.8 ± 2.0 mg N L−1. Increasing the nitrogen loading rate from 52.4 to 104.8 g N m−3 d−1 with stepwise decreasing HRT from 32 to 16 h resulted in an average TN removal efficiency of 81% without nitrite accumulation. The average observed NO3−–N (residue)/NH4+–N (consumed) ratio of 8% was below the “theoretical ratio” of 13% and further reduction of nitrate residue needs to be addressed. Fluorescence in situ hybridization (FISH) and high-throughput sequencing analyses showed the coexistence of anammox bacteria and ammonium-oxidizing bacteria (AOB) in both biofilm and granular samples. Anammox bacteria accounted for up to 63.3% of the microbial community of the granules, with Candidatus Jettenia being the distinctly dominant anammox genus. In contrast, the biofilm contained abundant Nitrosomonadaceae (AOB, 33.1%). In addition, the brown–yellow granules exhibited a more balanced community structure with anammox bacteria and AOB accounting for 33.7% and 18.2%, respectively, which may contribute to the long-term operation of single-stage nitritation–anammox process. These results demonstrate that the nitritation–anammox UMABR could potentially be used for nitrogen removal from mainstream in some specific regions with relatively warm temperature.

Deciphering the evolution of the functional genes and microbial community of the combined partial nitritation-anammox process with nitrate build-up and its in situ restoration

The operation of a combined partial nitritation-anammox (CPNA) process was hampered by the overgrowth of NOB, which was then successfullyin siturestored by the combination of hydroxylamine (NH2OH) dosing and sludge retention time (SRT) control.

Greenhouse Gases Emissions from Wastewater Treatment Plants: Minimization, Treatment, and Prevention

The operation of wastewater treatment plants results in direct emissions, from the biological processes, of greenhouse gases (GHG) such as carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O), as well as indirect emissions resulting from energy generation. In this study, three possible ways to reduce these emissions are discussed and analyzed:(1)minimization through the change of operational conditions,(2)treatment of the gaseous streams, and(3)prevention by applying new configurations and processes to remove both organic matter and pollutants. In current WWTPs, to modify the operational conditions of existing units reveals itself as possibly the most economical way to decrease N2O and CO2emissions without deterioration of effluent quality. Nowadays the treatment of the gaseous streams containing the GHG seems to be a not suitable option due to the high capital costs of systems involved to capture and clean them. The change of WWTP configuration by using microalgae or partial nitritation-Anammox processes to remove ammonia from wastewater, instead of conventional nitrification-denitrification processes, can significantly reduce the GHG emissions and the energy consumed. However, the area required in the case of microalgae systems and the current lack of information about stability of partial nitritation-Anammox processes operating in the main stream of the WWTP are factors to be considered.

Predation of nitritation-anammox biofilms used for nitrogen removal from wastewater.

Predation is assumed to be a major cause of bacterial mortality in wastewater treatment plants (WWTP). Grazing on the slowly growing autotrophic ammonia oxidizing bacteria (AOB) and anaerobic ammonium oxidizing bacteria (AMX) may result in loss of biomass, which could compromise nitrogen removal by the nitritation-anammox process. However, predation, particularly of anaerobic AMX, is unknown. We investigated the presence of protozoa, AOB and AMX and the possible predation in nitritation-anammox biofilms from several WWTPs. By fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM), predator and prey were localized in intact biofilm cryosections. Different broad morphological types of protozoa were found at different biofilm depths. Large variations in abundance of protozoa were seen. One reactor showed a predation event of amoeba-like protozoa, forming grazing fronts reaching deep biofilm regions that were dominated by the anaerobic AMX. Both AOB and AMX were grazed by the amoeba, as revealed by FISH-CLSM. Hence, even AMX, living in the deeper layers of stratified biofilms, are subjected to predation. Interestingly, different colocalization was observed between the amoeba-like protozoa and two different Ca. Brocadia AMX sublineages, indicating different grazing patterns. The findings indicate that predation pressure can be an important factor regulating the abundance of AOB and AMX, with implications for nitrogen removal from wastewater.

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.

Removal of reverse-fluxed ammonium by anammox in a forward osmosis system using ammonium bicarbonate as a draw solute

Reverse flux of ammonium draw solute is a serious problem for applying forward osmosis (FO) in water/wastewater treatment. In this study, anaerobic ammonium oxidization (anammox) was synergistically linked to FO for removal of reverse-fluxed ammonium, thereby creating an osmotic anammox system. The feasibility of this system was demonstrated through both batch and continuous operation, and the anammox process was developed in two stages: sole anammox and nitritation–anammox. With addition of nitrite, the sole anammox process achieved an effluent ammonium concentration of 9.9±9.5mgNL−1. The nitritation–anammox maintained an ammonium concentration of 3.1±4.2mgNL−1, and increased the water flux to 2.46±0.24LMH (Lm−2h−1) compared with the sole anammox (1.90±0.14LMH). The nitritation–anammox process exhibited advantages over anammox process in assisting the FO with respect to water flux improvement and chemical savings. The osmotic anammox system can be linked to previously developed microbial electrolysis cells that recover ammonium from high-strength wastes as a draw solute for FO operation. The results encourage further investigation of this system for effects of organic residues, decreasing nitrate accumulation, understanding biofilm on the FO membrane, and long-term performance with actual waste.

Nitrogen removal performance and microbial distribution in pilot- and full-scale integrated fixed-biofilm activated sludge reactors based on nitritation-anammox process

Nitritation–anammox process was successfully established in pilot- and full-scale integrated fixed-film activated sludge (IFAS) reactors. An average nitrogen removal efficiency of 80% was achieved under ammonium loading rate of 0.7–1.3kgN/(m3d) in the pilot-scale reactor (12m3). Moreover, molecular analysis showed that ammonium oxidizing bacteria (AOB) were more abundant in the activated sludge while anammox bacteria were primarily located in the biofilm. The segregation of AOB and anammox bacteria enhanced the nitrogen removal rate and operational stability. Furthermore, a full-scale IFAS reactor of 500m3 was set-up to treat sludge dewatering liquors. An average nitrogen removal efficiency of 85% and a nitrogen removal rate of 0.48kgN/(m3d) were achieved after inoculation. It was noted that high influent suspended solids would seriously affect the performance of the IFAS system. Therefore, a pre-treatment was proposed to reduce suspended solid in the full-scale application.

Development of the combined nitritation–anammox process in an upflow anaerobic sludge blanket (UASB) reactor with anammox granules

The combined nitritation–anaerobic ammonium oxidation (anammox) process represents a promising innovative technology for biological nitrogen removal, especially for treating wastewater with low chemical oxygen demand (COD) to ammonium ratios. In the present study, a laboratory-scale upflow anaerobic sludge blanket (UASB) reactor, which has been operated under low oxygen levels (⩽0.5mgL−1) for over 280days, was shown to remove about 88% of the supplied total nitrogen loads without nitrite/nitrate accumulation. During the start-up period, the reactor was initially inoculated with enriched anammox granules and fed with synthetic wastewater, where the ammonium concentration was fixed but nitrite concentration was gradually decreased to zero. As the influent NO2−-N/NH4+-N ratio decreased, O2 supply was increased gradually to facilitate the conversion of ammonium to nitrite by ammonium oxidation bacteria (AOB). Our results showed that the nitritation–anammox granules with porous structures were enriched and featured good settling ability. Microbial community composition was investigated by fluorescence in situ hybridization (FISH), which demonstrated the coexistence of AOB and anammox bacteria in the granules. The two groups of bacteria exhibited an overlapping growth style, which can improve ammonium availability to anammox bacteria and facilitate the immediate consumption of nitrite (produced by AOB) by anammox bacteria.

In-situ restoring nitrogen removal for the combined partial nitritation-anammox process deteriorated by nitrate build-up

The nitrogen removal performance in the combined partial nitritation-anammox (CPNA) process was seriously deteriorated by the nitrate build-up. The purpose of this study was to develop and optimize an in-situ restoring strategy based on hydroxylamine (NH2OH) dosing and solids retention time (SRT) control for the deteriorated CPNA process. Results showed that the 0.55kgNm−3d−1 of nitrogen removal rate could be recovered by 20mgNH2OHL−1 of hydroxylamine dosing and 40 days of SRT control, the nitrate concentration in effluent was decreased from the highest 548.4mgNL−1 during deterioration to 65.1mgNL−1 after restoration, and the ratio of NO3−-Nproduced/NH4+-Nconsumed in one SBR cycle was reduced from the highest 87.0% to 9.13% finally. The inhibition of nitrite-oxidizing bacteria (NOB) by NH2OH dosing alone was reversible because the nitrate build-up occurred again from 106.9 to 287.6mgNL−1 within just 11 days after NH2OH dosing was stopped. The evolution of the anammox bacteria, ammonium-oxidizing bacteria (AOB) and NOB from quantitative PCR (qPCR) assays verified the changes of the nitrogen removal performance of the CPNA process and proved that this in-situ restoration strategy could successfully solve the problem of nitrate build-up in the CPNA process.

The performance of a combined nitritation–anammox reactor treating anaerobic digestion supernatant under various C/N ratios

A combined nitritation–anammox reactor was developed to treat the digestion supernatant under various C/N ratios. Due to the difficulties for heterotroph to utilize the refractory organics, the reactor presented relatively stable performance with increasing supernatant addition. Nevertheless, the adverse effects of supernatant would accumulate during the long-term operation and thus weakened the activity and shock resistance of microbes, which further led to the gradual decrease of reactor performance after 92days' operation. Under this circumstance, supernatant with volatile fatty acids (VFAs) residuals was further introduced into the reactor to investigate the performance of combined nitritation–anammox process with VFA addition. With the appearance of VFAs, the nitrogen removal performance gradually restored and the reactor finally achieved stable and efficient performance with C/N ratio of 0.35. The VFA residuals within 150mg/L in the supernatant served as the extra electron donors and stimulated the heterotrophic denitrification process, which was vital for the enhancement of reactor. The nitrogen removal rate and total nitrogen removal efficiency reached 0.49kgN/(m3·day) and 88.8% after 140days' operation, respectively. The combined nitritation–anammox reactor was proved suitable to treat digestion supernatant.

The effect of inorganic carbon on microbial interactions in a biofilm nitritation–anammox process

The overarching goal of this study was to determine the role of inorganic carbon (IC) in influencing the microbial ecology, performance and nitrogen turnover by individual microbial communities of a biofilm based combined nitritation–anammox process. IC limitation was transiently imposed by reducing the IC input from 350% to 40% of the stoichiometric requirement for 40 days. The principal impact observed during IC limitation was the overgrowth of nitrite oxidizing bacteria (NOB) at the expense of anaerobic ammonia oxidizing bacteria (AMX). On the other hand, the concentrations of ammonia oxidizing bacteria (AOB) were relatively stable during the imposition of and recovery from IC limitation. The resulting dominance of NOB, in terms of their concentration and contribution to nitrite consumption over AMX, resulted, in turn, in a decrease in overall nitrogen removal from 78 ± 2.0% before IC limitation to 46 ± 2.9% during IC limitation. Upon recovery back to non-limiting IC input, it took an inordinately long time (about 57*HRT) for the N-removal to recover back to pre-limitation conditions. Even after recovery, NOB were still persistent in the biofilm and could not be washed out to pre-limitation concentrations. The emission of nitrous oxide (N2O) and nitric oxide (NO), likely from AOB, transiently increased in concert with transient increases in ammonia and hydroxylamine concentrations during the period of IC limitation. Therefore, an unintended consequence of IC limitation in nitritation–anammox systems can be an increase in their greenhouse gas footprint, in addition to compromised process performance. Most emphasis to date on nitritation and anammox studies has been on the nitrogen cycle. The results of this study demonstrate that the differing strategies used by AOB, NOB and AMX to compete for their preferred assimilative carbon source can also significantly influence the microbial ecology, performance and carbon footprint of such processes.

Innovative Approaches for Nitrogen Removal from Sewage: The Possible Use of Combined Partial Nitritation-Anammox Process

Innovative Approaches for Nitrogen Removal from Sewage: The Possible Use of Combined Partial Nitritation-Anammox Process

Microbial characterization of aggregates within a one-stage nitritation–anammox system using high-throughput amplicon sequencing

Abstract The combined nitritation–anammox process has recently been studied extensively from an engineering perspective. However, the importance of microbial communities of this process was generally underestimated. In this study, a lab-scale nitritation–anammox sequencing batch reactor (SBR) was established and the microbial community structure was further characterized, in order to provide the comprehensive insight into the key microbial groups in one-stage nitritation–anammox system. In general, a total nitrogen removal rate of 0.18 g N g −1 VSS d −1 was obtained after 180 days when the nitrogen loading rate was 0.5 kg N m −3 d −1 (hydraulic retention time of 1 d). Flexible flocs and compact granules were both found in the system, and this morphological differences were basically caused by the different microbial compositions, that is, flocs mainly consisted of filamentous bacteria and granules dominated by anammox bacteria. Results from high-throughput sequencing analysis revealed that Proteobacteria , Chloroflexi and Planctomycetes were selected and established a stable foothold in the community over the startup period, probably driven by the availability of substrate in the influent. Apart from nitrifiers of the Proteobacteria and anammox bacteria of the Planctomycetes , members of Chloroflexi constitute a large portion (>25%), which indicate that heterotrophs ( Chloroflexi ) survived by soluble microbial products (SMP) of autotrophs should not be neglected in the autotrophic system. This study could be useful for better understanding of one-stage nitritation–anammox system, especially for the interaction between autotrophs and heterotrophs in the system.

Differentiation in the microbial ecology and activity of suspended and attached bacteria in a nitritation-anammox process.

A directed differentiation between the biofilm and suspension was observed in the molecular microbial ecology and gene expression of different bacteria in a biofilm nitritation-anammox process operated at varying hydraulic residence times (HRT) and nitrogen loading rates (NLR). The highest degree of enrichment observed in the biofilm was of anaerobic ammonia-oxidizing bacteria (AMX) followed by that of Nitrospira spp. related nitrite-oxidizing bacteria (NOB). For AMX, a major shift from Candidatus "Brocadia fulgida" to Candidatus "Kuenenia stuttgartiensis" in both suspension and biofilm was observed with progressively shorter HRT, using discriminatory biomarkers targeting the hydrazine synthase (hzsA) gene. In parallel, expression of the hydrazine oxidoreductase gene (hzo), a functional biomarker for AMX energy metabolism, became progressively prominent in the biofilm. A marginal but statistically significant enrichment in the biofilm was observed for Nitrosomonas europaea related ammonia-oxidizing bacteria (AOB). In direct contrast to AMX, the gene expression of ammonia monooxygenase subunit A (amoA), a functional biomarker for AOB energy metabolism, progressively increased in suspension. Using gene expression and biomass concentration measures in conjunction, it was determined that signatures of AOB metabolism were primarily present in the biofilm throughout the study. On the other hand, AMX metabolism gradually shifted from being uniformly distributed in both the biofilm and suspension to primarily the biofilm at shorter HRTs and higher NLRs. These results therefore highlight the complexity and key differences in the microbial ecology, gene expression and activity between the biofilm and suspension of a nitritation-anammox process and the biokinetic and metabolic drivers for such niche segregation.

Response of Different Nitrospira Species To Anoxic Periods Depends on Operational DO

The exploitation of a lag phase in nitrate production after anoxic periods is a promising approach to suppress nitrite oxidizing bacteria, which is crucial for implementation of the combined partial nitritation-anammox process. An in-depth study of the actual lag phase in nitrate production after short anoxic periods was performed with varied temperatures and air flow rates. In monitored batch experiments, biomass from four different full-scale partial nitritation-anammox plants was subjected to anoxic periods of 5-60 min. Ammonium and the nitrite that was produced were present to reproduce reactor conditions and enable ammonium and nitrite oxidation at the same time. The lag phase observed in nitrite oxidation exceeded the lag phase in ammonium oxidation after anoxic periods of more than 15-20 min. Lower temperatures slowed down the conversion rates but did not affect the lag phases. The operational oxygen concentration in the originating full scale plants strongly affected the length of the lag phase, which could be attributed to different species of Nitrospira spp. detected by DGGE and sequencing analysis.