Partial Nitritation And Anammox Research Articles

Activation of iron oxides through organic matter-induced dissolved oxygen penetration depth dynamics enhances iron-cycling driven ammonium oxidation in microaerobic granular sludge

The iron redox cycle can enhance anammox in treating low-strength ammonia wastewater. However, maintaining an effective iron redox cycle and suppressing nitrite-oxidizing bacteria in a one-stage partial nitritation and anammox (PN/A) process poses challenges during long-term aeration. We proposed a novel and simple strategy to achieve an efficient iron redox cycle in an iron-mediated anoxic-microaerobic (A/O) process by controlling organic matter (OM) at medium-strength levels (30–110 mg COD/L) in microaerobic granular sludge (MGS)-dominated reactor. The developed A/O process consistently achieved >90 % OM removal and >75 % nitrogen removal. Medium-strength OM varied the penetration depths of dissolved oxygen (DO) in MGS, regulating redox conditions and promoting redox reactions across MGS layers, thus activating accumulated inert iron oxides. Ammonia-oxidizing bacteria (Nitrosomonas), iron-reducing bacteria (e.g., Ignavibacterium, Geobacter), and anammox bacteria (Ca. Kuenenia) coexisted harmoniously in MGS. This coexistence ensured high anammox and Feammox rates along with a robust iron redox cycle, thereby mitigating the adverse impacts of fluctuating DO and OM on one-stage PN/A process stability. The identification of iron reduction-associated genes within Ca. Kuenenia, Ignavibacterium, and Geobacter suggests their potential roles in supporting Feammox coupled in one-stage PN/A process. This study introduces an iron-cycle-driven A/O process as an energy-efficient alternative for simultaneous carbon and nitrogen removal from low-strength wastewater.

Full-Scale Demonstration of Nitrogen Removal from Mature Landfill Leachate Using a Two-Stage Partial Nitritation and Anammox Process

The excessive discharge of nitrogen leads to water eutrophication. The partial nitritation and anammox (PN/A) process is a promising technology for biological nitrogen removal in wastewater treatment. However, applying it to mature landfill leachate (MLL) faces challenges, as the toxic substances (e.g., heavy metal) within MLL inhibit the activity of anammox bacteria. Therefore, most previous studies focused on diluted, pretreated, or chemically adjusted MLL. This study demonstrated at full scale that the two-stage PN/A process can treat raw MLL. Initially, the operational issue of sludge floatation resulted in rapid biomass loss with overflow discharging, which selectively suppresses nitrite-oxidizing bacteria (NOB), promoting the achievement of nitrite accumulation. After that, the NOB suppression was self-sustained by the high in situ free ammonia concentration, i.e., 26.2 ± 15.9 mg N/L. In the subsequent anammox tank, nitrogen removal primarily occurred via the anammox process, complemented by denitrification, achieving total nitrogen removal efficiency exceeding 72%. In addition, the nitrogen removal capacity of this system was significantly influenced by temperature with the nitrogen-loading rate above 0.4 kg N/m3/d at 38 °C and approximately 0.1 kg N/m3/d at 21 °C. The optimization of system operation, such as gradually increasing MLL content, remains necessary to enhance nitrogen removal capacity further.

Dynamic pulse approach to enhancing mainstream Anammox process stability: Integrating sidestream support and tackling nitrite-oxidizing bacteria challenges

Nitrite-oxidizing bacteria (NOB) seriously threaten the partial nitritation and Anammox (PN/A) process, hindering its mainstream application. Herein, a one-stage PN/A reactor was continuously operated for 245 days under nitrogen loading rate lifted from 0.4 g N/L/d to 0.6 g N/L/d and 0.8 g N/L/d with the nitrogen removal efficiency of 71 %, 64 %, and 41 %, respectively. Furthermore, the NOB species over time was identified as Nitrospira_sp._OLB3, exhibiting an increase of the relative abundance from 0.9 % to 4.3 %. The hydroxyapatite (HAP) granules gradually lost their microbiological function of Anammox bacteria then aged, leading to NOB dominance. Therefore, one “pulse therapy” was introduced and combined with “continuous enhancement” of Anammox sludge supported by sidestream to competitively limit the NOB dynamics. The treatment's effect persisted for around two months. The strategy that returning at least 50 % of the impaired HAP granular sludge to the sidestream for recultivation could fulfill the bottlenecks of mainstream PN/A.

Decrease in Oxygen Concentration for the Fast Start-Up of Partial Nitritation/Anammox without Inoculum Addition

Initiating the partial nitritation and anammox (PN/A) process without inoculation poses a significant challenge. Thus, there is a notable amount of interest in devising a straightforward strategy for the start-up of PN/A. This study demonstrates the feasibility of achieving the rapid start-up of a one-stage PN/A process within a moving-bed sequencing batch biofilm reactor (MBSBBR) by reducing the oxygen concentrations: 3.0 mg O2/L (Stage I), 2.0 mg O2/L (Stage II), and 1.0 mg O2/L (Stage III). The anammox activity was observed 15 days after a gradual decrease in the oxygen concentration and confirmed using a specific anammox activity test (5.9 mg N/gVSS∙h). During Stage III, the average total inorganic nitrogen (TIN) removal efficiency was 60.6%. The relative abundance of planctomycetes, a typical phylum representing anammox microorganisms, increased almost three times from Stage I (2.8%) to Stage III (7.1%). These results demonstrate the potential of a decrease in oxygen concentration for the fast start-up of the one-stage partial nitritation and anammox process without inoculation. The implementation of the studied DO strategy has practical implications for wastewater treatment plant operators, particularly in the start-up of the PN/A processes. Additionally, batch assays allow for the rapid assessment of treatment plant performance, providing real-time insights into its functionality and, thereby, optimizing wastewater treatment practices.

Sustainable mainstream deammonification by ion exchange and bioregeneration via partial nitritation/anammox

The partial nitritation and anammox (PN/A) process has gained popularity for the treatment of nitrogen removal in wastewater due to significant energy savings and its potentially much lower CO2 footprint. However, the treatment of mainstream municipal wastewater by PN/A has been limited mainly due to its unsuitable composition. In this research, we apply ion exchange using a zeolite column to selectively remove and concentrate ammonium from mainstream municipal wastewater. After an absorption phase, the ion exchange column is regenerated using a brine solution. The ammonium rich brine is “bioregenerated” in a PN/A reactor where ammonium is converted to nitrogen gas allowing the brine to be reused in another cycle of ion exchange regeneration. To successfully remove ammonium from the spent brine, anammox and ammonia oxidizing bacteria (AOB) were first cultivated in separate reactors under hypersaline conditions (4.0 %) and later combined in a single PN/A reactor. After continuous operation with sea water, the PN/A reactor treated recirculating brine from the ion exchange column for 48 cycles of ammonium absorption and bioregeneration with minimal blowdown. The various cations of the regenerant solution were stable except for calcium that reached very high values upwards of 3000 mg/L as Ca2+ and finally caused PN/A reactor failure due to mineral precipitation. The buildup of high concentrations of calcium in the regenerant was addressed in two ways: 1) 20 % regenerant replacement per cycle, and 2) precipitation of CaCO3 via the addition of sodium carbonate. Both methods were applied to 30 absorption and bioregeneration cycles each and shown to be effective in keeping calcium concentrations from accumulating in the regenerant allowing for stable PN/A reactor operation.

Effects of ammonia nitrogen and organic carbon availability on microbial community structure and ecological interactions in a full-scale partial nitritation and anammox (PN/A) system

Nutrient availability significantly impacts microbial biosynthesis, cell growth, and cell cycle progression. In this study, a full-scale plug-flow partial nitritation/anammox (PN/A) system was used to investigate variations in the microbial community structure in both immobilized carriers and flocs, as well as a gradual decrease in nutrient availability from upstream to downstream. We found that reduced ammonia nitrogen (from 150.4 to 30.6 mg/L) and organic carbon (from 415.7 to 342.8 mg/L) availability significantly lowered microbial diversity and altered microbial communities in biofilms other than flocs from upstream to downstream. The abundance of all anammox bacteria increased by 1.97 times, from 3.25×1010 to 6.40×1010 copies per gram of wet sludge, in the biofilm core microbiome. Furthermore, from upstream to downstream, taxa with lower ribosomal RNA operon copy numbers were consistently enriched in both biofilm and floc communities, indicating that slow-growing microorganisms are more likely to be enriched in low-nutrient environments. Rare taxa with a relative abundance of less than 0.1% exhibited unique metabolic functions, including amino acid, carbohydrate, cofactor, and vitamin metabolisms, which was inferred by PICRUST2 and persisted across the nutrient gradient in both the biofilm and floc communities. Despite their low abundance, they may play important roles in mediating the stability and function of the PN/A system. Overall, the results demonstrate the impact of a naturally formed ammonia nitrogen and organic carbon gradient in a full-scale plug-flow PN/A installation on nutrient availability and its effects on microbial diversity, community composition, and microbial interactions, which expands our fundamental understanding of this energy-efficient and promising biotechnology for treating high-strength ammonium wastewater.

Full-scale partial nitritation and anammox (PN/A) application in removing nitrogen from industrial wastewater: Performance and life cycle assessment

Anaerobic ammonium oxidation (anammox) is an innovative nitrogen removal process that is widely used in wastewater treatment due to its energy-saving potential and ability to reduce carbon emissions. However, research on the application of anammox in full-scale industrial wastewater treatment plants (WWTPs) is limited, and its environmental impacts are yet to be fully understood. This study evaluates the performance and environmental burdens of one-stage partial nitritation and anammox (PN/A) in a full-scale WWTP treating coal chemical wastewater, with different demonstration periods. Compared with conventional nitrification–denitrification, the energy footprint and carbon footprint of the PN/A process were significantly lower and effluent quality was improved. Based on the four-quadrant clustering method, the overall performance of the studied WWTP achieved the greatest synergy during the stable operation period. Life cycle assessment has shown that the PN/A process significantly reduces the system’s contribution to environmental indicators. According to the sensitivity analysis, the fluctuations in energy consumption and chemical usage have significant environmental impacts, with differential sensitivity in the environmental impacts of two demonstration periods. Therefore, considering the specific situation of the WWTPs, optimizing the parameters/indicators most sensitive to environmental impact is essential to reducing environmental loads. Importantly, this work demonstrated that PN/A can improve the operational performance of full-scale industrial WWTPs with lower environmental burdens, providing valuable guidance for the development of sustainable wastewater treatment processes.

Linking morphological features to anammox communities in a partial nitritation and anammox (PN/A) biofilm reactor

Partial nitritation/anammox (PN/A) has been recognized as a cost-efficient process for wastewater nitrogen removal. The addition of carriers could help achieve biomass retention and enhance the treatment efficiency by forming the dense biofilm. However, accurately determining the abundance of anammox bacteria (AnAOB) to evaluate the biofilm development still remains challenging in practice without access to specialized facilities and experimental skills. In this study, we explored the feasibility of utilizing the morphological features of anammox biofilm as an indication of the biofilm development progression, and its correlation with microbial communities was also revealed. The time-series biofilms from an integrated fixed-film activated sludge (IFAS) system with stable PN/A performance were sampled representing the different biofilm development stages. The biofilm morphological features including color and texture were respectively quantified by red (R) coordinate and Local binary pattern (LBP) descriptor via image processing. Hierarchy clustering analysis proved that the extracted morphological descriptors could well distinguish the different stages (colonization, succession, and maturation) of biofilm development. The microbial community dynamics of time-series anammox biofilms were investigated using the amplicon sequence variant (ASV) analysis. Candidatus Brocadia, as the typical AnAOB, dominated in the whole communities of 16.3%–20.0%, moreover, the biofilm development was found to be driven by distinct Brocadia species. Linear regression evidenced that the Brocadia abundance could be directly correlated to the value of R and LBP, and the total variation of microbial communities could be significantly explained by the morphological features via redundancy analysis. This study demonstrates a new way to monitor the biofilm development by extracting the visible features of anammox aggregates, which can help facilitate the automated control of anammox-based bioprocess.

Toward Energy Neutrality: Novel Wastewater Treatment Incorporating Acidophilic Ammonia Oxidation.

Chemically enhanced primary treatment (CEPT) followed by partial nitritation and anammox (PN/A) and anaerobic digestion (AD) is a promising roadmap to achieve energy-neutral wastewater treatment. However, the acidification of wastewater caused by ferric hydrolysis in CEPT and how to achieve stable suppression of nitrite-oxidizing bacteria (NOB) in PN/A challenge this paradigm in practice. This study proposes a novel wastewater treatment scheme to overcome these challenges. Results showed that, by dosing FeCl3 at 50 mg Fe/L, the CEPT process removed 61.8% of COD and 90.1% of phosphate and reduced the alkalinity as well. Feeding by low alkalinity wastewater, stable nitrite accumulation was achieved in an aerobic reactor operated at pH 4.35 aided by a novel acid-tolerant ammonium-oxidizing bacteria (AOB), namely, Candidatus Nitrosoglobus. After polishing in a following anoxic reactor (anammox), a satisfactory effluent, containing COD at 41.9 ± 11.2 mg/L, total nitrogen at 5.1 ± 1.8 mg N/L, and phosphate at 0.3 ± 0.2 mg P/L, was achieved. Moreover, the stable performances of this integration were well maintained at an operating temperature of 12 °C, and 10 investigated micropollutants were removed from the wastewater. An energy balance assessment indicated that the integrated system could achieve energy self-sufficiency in domestic wastewater treatment.

Unveiling the effects of soluble starch, ethanol, and sodium acetate on the interactions of functional microorganisms and nitrogen removal in a partial nitritation and anammox biofilm system

Different organic matters have different effects on the nitrogen removal performance of partial nitritation and anammox (PN/A) biofilm system. In this study, the effects of adding soluble starch, ethanol, and sodium acetate on the functional microorganisms and nitrogen removal in the PN/A biofilm system are investigated. The results show that ethanol had the strongest effects on the ammonium-oxidizing bacteria (AOB) growth, enrichment, and ammonium nitrogen removal rate, followed by soluble starch and sodium acetate. However, the suppression of anaerobic ammonium oxidation bacteria (AnAOB) and decrease in total nitrogen removal rate were affected the most by ethanol, followed by soluble starch and sodium acetate. In addition, the enrichment of nitrifying oxidizing bacteria (NOB) was the most strongly influenced by ethanol, followed by soluble starch and sodium acetate. Furthermore, the enrichment of phylum Proteobacteria was facilitated by ethanol > soluble starch > sodium acetate. The enrichment of phylum Nitrospirae and genus Nitrospira were influenced by ethanol > soluble starch > sodium acetate. However, the suppression of the phylum Planctomycetes and the genus Candidatus Brocadia were affected by ethanol > soluble starch > sodium acetate. These results show that the PN/A biofilm system is suitable for the treatment of ammonium nitrogen wastewaters containing organic salt (sodium acetate) but not for treating wastewaters containing organic alcohol (ethanol) and macromolecular organic matter (soluble starch).

Ammonium concentration determines oxygen penetration depth to impact the suppression of nitrite-oxidizing bacteria inside partial nitritation and anammox biofilms

Biofilm-based Partial Nitritation and Anammox (PN/A) process has gained wide popularity in treating high ammonium (∼1000 mg NH4+-N/L) sidestream wastewater while facing a significant challenge in terms of nitrite-oxidizing bacteria (NOB) suppression when applied for low-strength municipal wastewater treatment with low ammonium concentration (∼50 mg N/L). Here, the significance of residual ammonium (NH4+) concentration to the mainstream PN/A biofilm system was systematically investigated through long-term and short-term experiments, in conjunction with analysis of the stratification of functional microorganisms and measurement of oxygen penetration. The experimental results collectively showed that a residual NH4+ concentration of ∼ 50 mg N/L was essential to stable NOB suppression, while a lower concentration of ∼ 5 mg N/L undesirably caused NOB proliferation. This result suggests that low NH4+ concentration in mainstream wastewater would lead to the intrinsic difficulty of NOB suppression in a biofilm PN/A process. Moreover, this study reveals that high NH4+ concentration promotes the activity of ammonia oxidizing bacteria (AOB) occupying the biofilm surface, thereby reducing oxygen penetration depth. Accordingly, NOB that inhabit a deep region of biofilm are suppressed by the limiting oxygen, while anammox bacteria are favoured instead. These findings provide important knowledge for the process control and expand the understanding of microbial interaction in mainstream PN/A biofilm systems.

Long-term effect of zero-valent iron on one-stage partial nitritation and anammox

Partial nitritation and anammox (PN/A) is an energy- and carbon-saving process for nitrogen removal from organic deficient wastewater. However, the PN/A effluent often requires polishing to eliminate the nitrate produced by anammox. Iron as the essential element for living organisms has been tested for nitrate reduction and phosphorus recovery in wastewater treatment processes. Here, we proposed a novel process coupling PN/A with iron-based denitrification for efficiently removing nitrogen and phosphorus in a single reactor. Two laboratory-scale bioreactors were operated under oxygen-limited conditions in parallel. Fe0 with a dosage of ∼ 27 mg Fe/L-wastewater was continuously dosed in the experimental reactor, with the other serving as a control. Throughout 300 days of operation, the results showed that experimental system always had significantly high removal efficiencies of organic carbon, nitrogen and phosphorus compared with control system. The total nitrogen removal achieved 90.1 ± 1.2 % at the rate of 0.96 ± 0.03 kg N/(m3•d) due to the improved activity of ammonia oxidizers, anammox bacteria and denitrifiers and enriched nitrogen conversion pathway (i.e., iron-based autotrophic denitrification) with Fe0 supplement. The total phosphorus removal was synchronously improved to 80.2 ± 9.1 % mainly driven by the phosphate precipitation with iron. This study provides new insights into achieving high-level nitrogen and phosphorus removal in a single reactor, and deepens our understanding of the improved nutrients removal mechanism mediated by iron.

Long-term adaptation of two anammox granules with different ratios of Candidatus Brocadia and Candidatus Jettenia under increasing salinity and their application to treat saline wastewater

Nitrogen removal in saline wastewater is a challenge of the anaerobic ammonium oxidation (anammox) process, which is dominated by freshwater anammox bacteria (FAB). Candidatus Brocadia and Candidatus Jettenia, the most widely used FABs, have been separately applied and evaluated for their ability to treat saline wastewater. To understand the effect of salinity on nitrogen removal capability when they present together in an anammox granule, we compared two anammox granules: GRN1 was evenly dominated by Ca. Brocadia (42 %) and Ca. Jettenia (43 %), while GRN2 was dominated with mostly Ca. Brocadia (90 %) and a small amount of Ca. Jettenia (1 %). Each granule was inoculated into a continuous column reactor to treat artificial wastewater containing 150 mg NH4+-N/L and 150 mg NO2−-N/L under increasing saline conditions for 250 days. GRN1 showed superior and more stable nitrogen removal than GRN2 under saline conditions of up to 15 g NaCl/L. Under high-saline conditions, both the granules' sizes decreased (larger GRN1 than GRN2 in initial). The mass percent of Na salt increased (more in GRN2) and mineral contents decreased more in GRN1. High-throughput sequencing for microbial community analysis showed that Planctomycetes in GRN1 (85 %) and GRN2 (92 %) decreased to 14 % and 12 %, respectively. The ratio of Ca. Brocadia and Ca. Jettenia in GRN1 changed to 37 % and 63 %, respectively, whereas the ratio in GRN2 (99 % and 1 %, respectively) did not change. Both salt-adapted granules were applied to the two-stage partial nitritation and anammox (PN/A) process to treat high strength ammonium (400 mg/L) wastewater under high saline condition (15 g NaCl/L). The PN/A process containing GRN1 showed more stable nitrogen removal performance during approximately 100 days of operation. These results suggest that the anammox granules evenly dominated by two FABs, Ca. Brocadia and Ca. Jettenia, would be advantageous to treat high-strength NH4+ wastewater under high-saline conditions.

A novel vertical dual-loop reactor for rapid start-up of simultaneous partial nitrification and anammox process in treating landfill leachate: Performances and mechanisms

A novel vertical dual-loop reactor (VDLR) was developed to start and conduct a single-stage partial nitritation (PN) and anammox (PN/A) process for treating landfill leachate. Results showed that the total nitrogen (TN) removal reached 1.54 kg N/m3·d in the VDLR. It exhibited excellent mixing uniformity and buffer performance, which can increase the nitrogen removal performance up to 42.1 % via the improvement of anammox granular sludge activity (a particle size of 0.5–1 mm). Mass balance and microbial analysis indicated that the VDLR achieved efficient TN removal via anammox (99.24 %) and AOB (Nitrosomonas and Ellin6067) and anAOB (Candidatus kuenenia) played a vital role in this process.

Coexistence and competition of the nitrifying and anammox bacteria in SMBBR reactor with partial nitritation/anammox process treating synthetic low-strength ammonium wastewater

The nitrite-oxidizing bacteria (NOB) suppression largely restricts the implementation of partial-nitritation and anammox (PN/A) process for low-strength ammonium wastewater treatment. The main goal of the present study was to verify the feasibility of sequencing moving bed biofilm reactor (SMBBR) treating synthetic low-strength ammonium wastewater and the influence of operating conditions and microbial competitions on NOB suppression. The reactor was operated in sequencing batch reactor (SBR) mode with influent ammonium of 50 mg/L, in which the seeded biofilm harvested from an existing similar PN/A reactor treating high-ammonium synthetic wastewater. After increasing the dissolved oxygen (DO) from 0.50 to 1.25 mg/L-with hydraulic retention time (HRT) gradually decreasing from 15 to 5 h-stable nitrogen removal through PN/A was obtained. The long-term operation results showed nitrogen removal efficiencies of 66.23 ± 7.78 % were stably obtained at a relatively low filling ratio of 25 %. The reactor operated in SBR mode allowed NOB suppression and low effluent ammonium due to the ammonium gradient created in cycles. Further, the activity of NOB was controlled at a low level at medium DO concentration. Both ammonium-oxidizing bacteria (AOB) competition acting as DO-cut and anaerobic ammonium-oxidizing bacteria (anammox, AMX) competition acting as nitrite-sink were found to be essential for NOB suppression, and DO-cut played a dominated role. A simple hypothetical microstructure of one-stage PN/A biofilm, assuming AOB layered in the surface of the biofilm while AMX and NOB in the interior, was proposed to define the mechanism of DO-cut and Nitrite-sink. The obtained results demonstrated SMBBR could be regarded as an alternative guideline for successful operation of mainstream PN/A as compared to the integrated fixed film activated sludge (IFAS) systems.

A 20-Year Journey of Partial Nitritation and Anammox (PN/A): from Sidestream toward Mainstream.

Anaerobic ammonium oxidation (anammox) was discovered as a new microbial reaction in the late 1990s, which led to the development of an innovative energy- and carbon-efficient technology─partial nitritation and anammox (PN/A)─for nitrogen removal. PN/A was first applied to remove the nitrogen from high-strength wastewaters, e.g., anaerobic digestion liquor (i.e., sidestream), and further expanded to the main line of wastewater treatment plants (i.e., mainstream). While sidestream PN/A has been well-established with extensive full-scale installations worldwide, practical application of PN/A in mainstream treatment has been proven extremely challenging to date. A key challenge is achieving stable suppression of nitrite-oxidizing bacteria (NOB). This study examines the progress of NOB suppression in both sidestream- and mainstream PN/A over the past two decades. The successful NOB suppression in sidestream PN/A was reviewed, and these successes were evaluated in terms of their transferability into mainstream PN/A. Drawing on the learning over the past decades, we anticipate that a hybrid process, comprised of biofilm and floccular sludge, bears great potential to achieve efficient mainstream PN/A, while a combination of strategies is entailed for stable NOB suppression. Furthermore, the recent discovery of novel nitrifiers would trigger new opportunities and new challenges for mainstream PN/A.

Adaptation of anammox bacteria to low temperature via gradual acclimation and cold shocks: Distinctions in protein expression, membrane composition and activities

Anammox bacteria enable efficient removal of nitrogen from sewage in processes involving partial nitritation and anammox (PN/A) or nitrification, partial denitrification, and anammox (N-PdN/A). In mild climates, anammox bacteria must be adapted to ≤15 °C, typically by gradual temperature decrease; however, this takes months or years. To reduce the time necessary for the adaptation, an unconventional method of ‘cold shocks’ is promising, involving hours-long exposure of anammox biomass to extremely low temperatures. We compared the efficacies of gradual temperature decrease and cold shocks to increase the metabolic activity of anammox (fed batch reactor, planktonic “Ca. Kuenenia”). We assessed the cold shock mechanism on the level of protein expression (quantitative shot-gun proteomics, LCHRMS/MS) and the structure of membrane lipids (UPLCHRMS/MS). The shocked culture was more active (0.66±0.06 vs 0.48±0.06 kg-N/kg-VSS/d) and maintained the relative content of N-respiration proteins at levels consistent levels with the initial state, whereas the content of these proteins decreased in gradually acclimated culture. Cold shocks also induced a more efficient expression of potential cold shock proteins (e.g. ppiD, UspA, pqqC), while putative cold shock proteins CspB and TypA were upregulated in both cultures. Ladderane lipids characteristic for anammox evolved to a similar end-point in both cultures; this confirms their role in anammox bacteria adaptation to cold and indicates a three-pronged adaptation mechanism (ladderane alkyl length, introduction of shorter non-ladderane alkyls, polar headgroup). Overall, we show the outstanding potential of cold shocks for low-temperature adaptation of anammox bacteria and provide yet unreported detailed mechanisms of anammox adaptation to low temperatures.

Effective restoration of partial nitritation and anammox biofilm process by short-term hydroxylamine dosing: Mechanism and microbial interaction

The one-stage partial nitritation and anammox (PN-A) process frequently experiences deterioration from ammonium accumulation and nitrate build-up. In this study, hydroxylamine was dosed to restore the process from deterioration in a continuously aerated PN-A sequencing biofilm batch reactor, and the impact of hydroxylamine on the metabolism of PN-A process was studied. PN-A process was totally restored in 5 days via 10 mg N·L-1 hydroxylamine dosing, reducing nitrate-produced/ammonium-removed ratio from 28.5% to less than 11.0%. hydroxylamine dosing promoted biological production of nitric oxide and nitrous oxide and reduced the production of nitrate in the PN-A process. This study advanced the understanding of the metabolism versatility of hydroxylamine and nitric oxide as well as their function in interaction between aerobic ammonium oxidation bacteria and anaerobic ammonium oxidation bacteria, and proposed the potential application of hydroxylamine dosing in ammonium-contained wastewater treatment.

Unravelling adaptation of nitrite-oxidizing bacteria in mainstream PN/A process: Mechanisms and counter-strategies

Stable suppression of nitrite-oxidizing bacteria (NOB) is still a major challenge for the implementation of partial nitritation and anammox (PN/A) in mainstream treatment. Despite numerous suppression strategies demonstrated, it is increasingly recognized that NOB could develop resistance to these strategies, threatening the long-term stability of the mainstream PN/A process. This study aims to understand adaption mechanisms and develop counter-strategies to overcome the adaptation. To this end, three previously-demonstrated suppression strategies, including NOB inactivation via side stream sludge treatment with free ammonia (FA), the use of low dissolved oxygen (DO), and the use of anammox to scavenge nitrite, were stepwise applied, over a period of 800 days, to a laboratory-scale reactor treating effluent from a high-rate activated sludge (HRAS) plant. FA sludge treatment alone sustained nitrite accumulation for about two months, after which NOB adaptation occurred causing PN to fail. The FA adaptation was induced by a shift in the NOB community from Nitrospira to Ca. Nitrotoga. The latter was found to have higher resistance to FA and also a higher maximum specific growth rate. Low DO at 0.2–0.4 mg O2 L−1 was then applied, in conjunction with FA treatment, which successfully eliminated Ca. Nitrotoga and re-established PN. However, new and unidentified NOB with a higher apparent oxygen affinity emerged in three months, again leading to PN failure. Lastly, as the third strategy for NOB suppression, anammox was introduced as an in-situ nitrite-scavenger. The combo-strategy delivered reliable NOB suppression with no further adaptation in the remaining experimental period (eight months). The resulted one-stage PN/A reactor achieved a nitrogen removal efficiency of 84.2 ± 5.37%. A control reactor, operated in parallel under the same conditions but without FA treatment, only achieved 10.4 ± 4.6% nitrogen removal, with anammox completely outcompeted by NOB in the last phase.

Fast start-up and enhancement of partial nitritation and anammox process for treating synthetic wastewater in a sequencing bath biofilm reactor: Strategy and function of nitric oxide

In this study, the partial nitritation and anammox (PN-A) process was initiated within 30 days in a sequencing batch biofilm reactor (SBBR) by employing pre non-aeration and post non-aeration with fixed aeration rates. The average ammonia removal efficiency (ARE), total nitrogen removal efficiency (TNRE) of 98.5 ± 1.5% and 89.5 ± 1.6% were achieved. By doubling aeration rate and agitation rate and adopting pre non-aeration, the TNRR was promoted from 0.135 ± 0.013 kg N·m−3·d−1 to 0.285 ± 0.015 kg N·m−3·d−1, obtaining an average ARE and TNRE of 97.5 ± 1.5% and 85.5 ± 2.6%. Nitric oxide might induce anaerobic ammonia oxidation bacteria (AnAOB) during the start-up stage, and could be an indicator for synergetic state between ammonia oxidation bacteria (AOB) and AnAOB. Lower nitrous oxide emission factor of 0.51% was obtained. The abundance of AOB, AnAOB and nitrite oxidation bacteria (NOB) accounted for 1.6%, 19.3% and 0.3%, respectively.