Metatranscriptomic Analysis Reveals Synergistic Activities of Comammox and Anammox Bacteria in Full-Scale Attached Growth Nitrogen Removal System.

Leveraging comammox Nitrospira and anammox bacteria for shortcut nitrogen removal can drastically lower the carbon footprint of wastewater treatment facilities by decreasing aeration energy, carbon, alkalinity, and tank volume requirements while also potentially reducing nitrous oxide emissions. However, their co-occurrence as dominant nitrifying bacteria is rarely reported in full-scale wastewater treatment. As a result, there is a poor understanding of how operational parameters, in particular, dissolved oxygen, impact their activity and synergistic behavior. Here, we report the impact of dissolved oxygen concentration (DO = 2, 4, 6 mg/L) on the microbial community's transcriptomic expression in a full-scale integrated fixed film activated sludge (IFAS) municipal wastewater treatment facility where nitrogen removal is predominantly performed by comammox Nitrospira and anammox bacterial populations. 16S rRNA transcript compositions revealed anammox bacteria and Nitrospira were significantly more active in IFAS biofilms compared to suspended sludge biomass. In IFAS biofilms, anammox bacteria significantly increased hzo expression at lower dissolved oxygen concentrations and this increase was highly correlated with the amoA expression levels of comammox bacteria. Interestingly, the genes involved in nitrite oxidation by comammox bacteria were significantly more upregulated, relative to the genes involved in ammonia oxidation with decreasing dissolved oxygen concentrations. Ultimately, our findings suggest that comammox Nitrospira supplies anammox bacteria with nitrite via ammonia oxidation and that this synergistic behavior is dependent on dissolved oxygen concentrations.

Co-Occurrence and Cooperation between Comammox and Anammox Bacteria in a Full-Scale Attached Growth Municipal Wastewater Treatment Process.

Cooperation between comammox and anammox bacteria for nitrogen removal has been recently reported in laboratory-scale systems, including synthetic community constructs; however, there are no reports of full-scale municipal wastewater treatment systems with such cooperation. Here, we report intrinsic and extant kinetics as well as genome-resolved community characterization of a full-scale integrated fixed film activated sludge (IFAS) system where comammox and anammox bacteria co-occur and appear to drive nitrogen loss. Intrinsic batch kinetic assays indicated that majority of the aerobic ammonia oxidation was driven by comammox bacteria (1.75 ± 0.08 mg-N/g TS-h) in the attached growth phase, with minimal contribution by ammonia-oxidizing bacteria. Interestingly, a portion of total inorganic nitrogen (∼8%) was consistently lost during these aerobic assays. Aerobic nitrite oxidation assays eliminated the possibility of denitrification as a cause of nitrogen loss, while anaerobic ammonia oxidation assays resulted in rates consistent with anammox stoichiometry. Full-scale experiments at different dissolved oxygen (DO = 2 - 6 mg/L) setpoints indicated persistent nitrogen loss that was partly sensitive to DO concentrations. Genome-resolved metagenomics confirmed the high abundance (relative abundance 6.53 ± 0.34%) of two Brocadia-like anammox populations, while comammox bacteria within the Ca. Nitrospira nitrosa cluster were lower in abundance (0.37 ± 0.03%) and Nitrosomonas-like ammonia oxidizers were even lower (0.12 ± 0.02%). Collectively, our study reports for the first time the co-occurrence and cooperation of comammox and anammox bacteria in a full-scale municipal wastewater treatment system.

Mainstream short-cut N removal modelling: current status and perspectives.

This work gives an overview of the state-of-the-art in modelling of short-cut processes for nitrogen removal in mainstream wastewater treatment and presents future perspectives for directing research efforts in line with the needs of practice. The modelling status for deammonification (i.e., anammox-based) and nitrite-shunt processes is presented with its challenges and limitations. The importance of mathematical models for considering N2O emissions in the design and operation of short-cut nitrogen removal processes is considered as well. Modelling goals and potential benefits are presented and the needs for new and more advanced approaches are identified. Overall, this contribution presents how existing and future mathematical models can accelerate successful full-scale mainstream short-cut nitrogen removal applications.

Status, Challenges, and Perspectives of Mainstream Nitritation-Anammox for Wastewater Treatment.

The nitritation-anammox process is an efficient and cost-effective approach for biological nitrogen removal, but its application in treating mainstream wastewater remains a great challenge. Mainstream nitritation-anammox processes could create opportunities for achieving energy self-sufficient, or energy-generating water resource recovery facilities. Significant advancements have been achieved via pilot- and full-scale trials to overcome the major obstacles under mainstream conditions, such as repression of nitrite-oxidizing bacteria, limiting the overgrowth of denitrifiers, and effective selection and retention of ammonia-oxidizing bacteria and anammox bacteria. This review paper intends to provide a detailed update of research progress on mainstream nitritation-anammox processes, discuss metabolic interactions, and examine major challenges and possible solutions towards the future development.

Startup of a Partial Nitritation-Anammox MBBR and the Implementation of pH-Based Aeration Control.

The single-stage deammonification moving bed biofilm reactor (MBBR) is a process for treating high strength nitrogen waste streams. In this process, partial nitritation and anaerobic ammonia oxidation (anammox) occur simultaneously within a biofilm attached to plastic carriers. An existing tank at the James River Treatment Plant (76 ML/d) in Newport News, Virginia was modified to install a sidestream deammonification MBBR process. This was the second sidestream deammonification process in North America and the first MBBR type installation. After 4 months the process achieved greater than 85% ammonia removal at the design loading rate of 2.4 g /m2·d (256 kg /d) signaling the end of startup. Based on observations during startup and process optimization phases, a novel pH-based control system was developed that maximizes ammonium removal and results in stable aeration and effluent alkalinity.

Methods for increasing the rate of anammox attachment in a sidestream deammonification MBBR.

Deammonification (partial nitritation-anammox) is a proven process for the treatment of high-nitrogen waste streams, but long startup time is a known drawback of this technology. In a deammonification moving bed biofilm reactor (MBBR), startup time could potentially be decreased by increasing the attachment rate of anammox bacteria (AMX) on virgin plastic media. Previous studies have shown that bacterial adhesion rates can be increased by surface modification or by the development of a preliminary biofilm. This is the first study on increasing AMX attachment rates in a deammonification MBBR using these methods. Experimental media consisted of three different wet-chemical surface treatments, and also media transferred from a full-scale mainstream fully nitrifying integrated fixed-film activated sludge (IFAS) reactor. Following startup of a full-scale deammonification reactor, the experimental media were placed in the full-scale reactor and removed for activity rate measurements and biomass testing after 1 and 2 months. The media transferred from the IFAS process exhibited a rapid increase in AMX activity rates (1.1 g/m(2)/day NH(4)(+) removal and 1.4 g/m(2)/day NO(2)(-) removal) as compared to the control (0.2 g/m(2)/day NH(4)(+) removal and 0.1 g/m(2)/day NO(2)(-) removal) after 1 month. Two out of three of the surface modifications resulted in significantly higher AMX activity than the control at 1 and 2 months. No nitrite oxidizing bacteria activity was detected in either the surface modified media or IFAS media batch tests. The results indicate that startup time of a deammonification MBBR could potentially be decreased through surface modification of the plastic media or through the transfer of media from a mature IFAS process.

Comparing methanol and glycerol as carbon sources for mainstream partial denitrification/anammox in an IFAS process.

This study explored the implementation of mainstream partial denitrification with anammox (PdNA) in the second anoxic zone of a wastewater treatment process in an integrated fixed film activated sludge (IFAS) configuration. A pilot study was conducted to compare the use of methanol and glycerol as external carbon sources for an IFAS PdNA startup, with a goal to optimize nitrogen removal while minimizing carbon usage. The study also investigated the establishment of anammox bacteria on virgin carriers in IFAS reactors without the use of seeding, and it is the first IFAS PdNA startup to use methanol as an external carbon source. The establishment of anammox bacteria was confirmed in both reactors 102 days after startup. Although the glycerol-fed reactor achieved a higher steady-state maximum ammonia removal rate because of anammox bacteria (1.6 ± 0.3 g/m2/day) in comparison with the methanol-fed reactor (1.2 ± 0.2 g/m2/day), both the glycerol- and methanol-fed reactors achieved similar average in situ ammonia removal rates of 0.39 ± 0.2 g/m2/day and 0.40 ± 0.2 g/m2/day, respectively. Additionally, when the upstream ammonia versus NOx (AvN) control system maintained an ideal ratio of 0.40-0.50 g/g, the methanol-fed reactor attained a lower average effluent TIN concentration (3.50 ± 1.2 mg/L) than the glycerol-fed reactor (4.43 ± 1.6 mg/L), which was prone to elevated nitrite concentrations in the effluent. Overall, this research highlights the potential for PdNA in IFAS configurations as an efficient and cost-saving method for wastewater treatment, with methanol as a viable carbon source for the establishment of anammox bacteria. PRACTITIONER POINTS: Methanol is an effective external carbon source for an anammox startup that avoids the need for costly alternative carbon sources. The methanol-fed reactor demonstrated higher TIN removal compared with the glycerol-fed reactor because of less overproduction of nitrite. Anammox bacteria was established in an IFAS reactor without seeding and used internally stored carbon to reduce external carbon addition. Controlling the influent ammonia versus NOx (AvN) ratio between 0.40 and 0.50 g/g allowed for low and stable TIN effluent conditions.

Comammox and unknown ammonia oxidizers contribute to nitrite accumulation in an integrated A-B stage process that incorporates side-stream EBPR (S2EBPR).

A novel integrated pilot-scale A-stage high rate activated sludge, B-stage short-cut biological nitrogen removal and side-stream enhanced biological phosphorus removal (A/B-shortcut N-S2EBPR) process for treating municipal wastewater was demonstrated with the aim to achieve simultaneous and carbon- and energy-efficient N and P removal. In this studied period, an average of 7.62 ± 2.17 mg-N/L nitrite accumulation was achieved through atypical partial nitrification without canonical known NOB out-selection. Network analysis confirms the central hub of microbial community as Nitrospira, which was one to two orders of magnitude higher than canonical aerobic oxidizing bacteria (AOB) in a B-stage nitrification tank. The contribution of comammox Nitrospira as AOB was evidenced by the increased amoB/nxr ratio and higher ammonia oxidation activity. Furthermore, oligotyping analysis of Nitrospira revealed two dominant sub-clusters (microdiveristy) within the Nitrospira. The relative abundance of oligotype II, which is phylogenetically close to Nitrospira_midas_s_31566, exhibited a positive correlation with nitrite accumulation in the same operational period, suggesting its role as comammox Nitrospira. Additionally, the phylogenetic investigation suggested that heterotrophic organisms from the family Comamonadacea and the order Rhodocyclaceae embedding ammonia monooxygenase and hydroxylamine oxidase may function as heterotrophic nitrifiers. This is the first study that elucidated the impact of integrating the S2EBPR on nitrifying populations with implications on short-cut N removal. The unique conditions in the side-stream reactor, such as low ORP, favorable VFA concentrations and composition, seemed to exert different selective forces on nitrifying populations from those in conventional biological nutrient removal processes. The results provide new insights for integrating EBPR with short-cut N removal process for mainstream wastewater treatment.

Revisiting the role of Acinetobacter spp. in side-stream enhanced biological phosphorus removal (S2EBPR) systems.

We piloted the incorporation of side-stream enhanced biological phosphorus removal (S2EBPR) with A/B stage short-cut nitrogen removal processes to enable simultaneous carbon-energy-efficient nutrients removal. This unique configuration and system conditions exerted selective force on microbial populations distinct from those in conventional EBPR. Interestingly, effective P removal was achieved with the predominance of Acinetobacter (21.5 ± 0.1 %) with nearly negligible level of known conical PAOs (Ca. Accumulibacter and Tetrasphaera were 0.04 ± 0.10 % and 0.47 ± 0.32 %, respectively). Using a combination of techniques, such as fluorescence in situ hybridization (FISH) coupled with single cell Raman spectroscopy (SCRS), the metabolic tracing of Acinetobacter-like cells exerted PAO-like phenotypic profiling. In addition, comparative metagenomics analysis of the closely related Acinetobacter spp. revealed the EBPR relevant metabolic pathways. Further oligotyping analysis of 16s rRNA V4 region revealed sub-clusters (microdiversity) of the Acinetobacter and revealed that the sub-group (oligo type 1, identical (100 % alignment identity) hits from Acinetobacter_midas_s_49494, and Acinetobacter_midas_s_55652) correlated with EBPR activities parameters, provided strong evidence that the identified Acinetobacter most likely contributed to the overall P removal in our A/B-shortcut N-S2EBPR system. To the best of our knowledge, this is the first study to confirm the in situ EBPR activity of Acinetobacter using combined genomics and SCRS Raman techniques. Further research is needed to identify the specific taxon, and phenotype of the Acinetobacter that are responsible for the P-removal.

Side-Stream Enhanced Biological Phosphorus Removal (S2EBPR) enables effective phosphorus removal in a pilot-scale A-B stage shortcut nitrogen removal system for mainstream municipal wastewater treatment.

While the adsorption/bio-oxidation (A/B) process has been widely studied for carbon capture and shortcut nitrogen (N) removal, its integration with enhanced biological phosphorus (P) removal (EBPR) has been considered challenging and thus unexplored. Here, full-scale pilot testing with an integrated system combining A-stage high-rate activated sludge with B-stage partial (de)nitrification/anammox and side-stream EBPR (HRAS-P(D)N/A-S2EBPR) was conducted treating real municipal wastewater. The results demonstrated that, despite the relatively low influent carbon load, the B-stage P(D)N-S2EBPR system could achieve effective P removal performance, with the carbon supplement and redirection of the A-stage sludge fermentate to the S2EBPR. The novel process configuration design enabled a system shift in carbon flux and distribution for efficient EBPR, and provided unique selective factors for ecological niche partitioning among different key functionally relevant microorganisms including polyphosphate accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs). The combined nitrite from B-stage to S2EBPR and aerobic-anoxic conditions in our HRAS-P(D)N/A-S2EBPR system promoted DPAOs for simultaneous internal carbon-driven denitrification via nitrite and P removal. 16S rRNA gene-based oligotyping analysis revealed high phylogenetic microdiversity within the Accumulibacter population and discovered coexistence of certain oligotypes of Accumulibacter and Competibacter correlated with efficient P removal. Single-cell Raman micro-spectroscopy-based phenotypic profiling showed high phenotypic microdiversity in the active PAO community and the involvement of unidentified PAOs and internal carbon-accumulating organisms that potentially played an important role in system performance. This is the first pilot study to demonstrate that the P(D)N-S2EBPR system could achieve shortcut N removal and influent carbon-independent EBPR simultaneously, and the results provided insights into the effects of incorporating S2EBPR into A/B process on metabolic activities, microbial ecology, and resulted system performance.

Sidestream bio-P and mainstream anammox in a BNR process with upstream carbon capture.

The integration of biological phosphorus removal (bio-P) and shortcut nitrogen removal (SNR) processes is challenging because of the conflicting demands on influent carbon: SNR allows for upstream carbon diversion, but this reduction of influent carbon (especially volatile fatty acids [VFAs]) prevents or limits bio-P. The objective of this study was to achieve SNR, either via partial nitritation/anammox (PNA) or partial denitrification/anammox (PdNA), simultaneously with biological phosphorus removal in a process with upstream carbon capture. This study took place in a pilot scale A/B process with a sidestream bio-P reactor and tertiary anammox polishing. Despite low influent rbCOD concentrations from the A-stage effluent, bio-P occurred in the B-stage thanks to the addition of A-stage WAS fermentate to the sidestream reactor. Nitrite accumulation occurred in the B-stage via partial denitrification and partial nitritation (NOB out-selection), depending on operational conditions, and was removed along with ammonia by the tertiary anammox MBBR, with the ability to achieve effluent TIN less than 2 mg/L. PRACTITIONER POINTS: A sidestream reactor with sufficient fermentate addition enables biological phosphorus removal in a B-stage system with little-to-no influent VFA. Enhanced biological phosphorus removal is not inhibited by intermittent aeration and is stable at a wide range of process SRTs. Partial nitritation and partial denitrification are viable routes to produce nitrite within an A/B process with sidestream bio-P, for downstream anammox in a polishing MBBR.

Two alanine aminotranferases link mitochondrial glycolate oxidation to the major photorespiratory pathway in Arabidopsis and rice.

The major photorespiratory pathway in higher plants is distributed over chloroplasts, mitochondria, and peroxisomes. In this pathway, glycolate oxidation takes place in peroxisomes. It was previously suggested that a mitochondrial glycolate dehydrogenase (GlcDH) that was conserved from green algae lacking leaf-type peroxisomes contributes to photorespiration in Arabidopsis thaliana. Here, the identification of two Arabidopsis mitochondrial alanine:glyoxylate aminotransferases (ALAATs) that link glycolate oxidation to glycine formation are described. By this reaction, the mitochondrial side pathway produces glycine from glyoxylate that can be used in the glycine decarboxylase (GCD) reaction of the major pathway. RNA interference (RNAi) suppression of mitochondrial ALAAT did not result in major changes in metabolite pools under standard conditions or enhanced photorespiratroy flux, respectively. However, RNAi lines showed reduced photorespiratory CO(2) release and a lower CO(2) compensation point. Mitochondria isolated from RNAi lines are incapable of converting glycolate to CO(2), whereas simultaneous overexpression of GlcDH and ALAATs in transiently transformed tobacco leaves enhances glycolate conversion. Furthermore, analyses of rice mitochondria suggest that the side pathway for glycolate oxidation and glycine formation is conserved in monocotyledoneous plants. It is concluded that the photorespiratory pathway from green algae has been functionally conserved in higher plants.

Nitrogen removal capacity and carbon demand requirements of partial denitrification/anammox MBBR and IFAS processes.

A pilot study was conducted to investigate the carbon demand requirements and nitrogen removal capabilities of two mainstream partial denitrification/anammox (PdNA) processes: a two-zone, moving bed biofilm reactor (MBBR) process and an integrated fixed-film activated sludge (IFAS) process. The first MBBR zone conducted PdNA, while the second operated as an anammox zone. Operation of the IFAS process was conducted in two phases. The first phase of the operation involved minor external carbon addition, while the second phase of the operation involved controlled external carbon addition. The MBBR process produced an average effluent TIN concentration and chemical oxygen demand (COD)/TIN ratio of 2.81 ± 1.21 mg/L and 2.42 ± 0.77 g/g. The average effluent TIN concentrations and COD/TIN ratios for the IFAS process were 4.07 ± 1.66 mg/L and 1.08 ± 0.38 g/g during phase 1 and 3.30 ± 0.96 mg/L and 2.18 ± 0.99 g/g during phase 2. Despite having relatively low and unstable partial denitrification (PdN) efficiencies, both mainstream PdNA processes exhibited low effluent TIN concentrations and carbon requirements compared to nitrification/denitrification. Successful operation of the PdNA IFAS process indicates that mainstream PdNA can be implemented with minimal capital costs in a water resource recovery facility's second anoxic zone. PRACTITIONER POINTS: Low effluent TIN concentrations can be maintained in mainstream PdNA MBBR and IFAS processes with low external carbon demand. MBBR and IFAS PdNA processes do not require consistent or high PdN efficiencies to maintain low effluent TIN concentrations. IFAS and MBBR PdNA processes exhibit similar TIN and NH3 removal efficiencies. PdNA can be implemented in a second anoxic zone, using IFAS technology for anammox retention, with minimal capital costs.

Startup strategies for mainstream anammox polishing in moving bed biofilm reactors.

This study evaluated startup strategies for mainstream polishing anammox moving bed biofilm reactors (MBBRs) without anammox bacterial (AMX) biomass inoculation. Two types of startups were tested: anammox only (no external carbon addition) and partial denitrification/anammox (PdNA) with glycerol addition. Reactors were started with either virgin carriers or carriers with a preliminary biofilm from a mainstream aerobic integrated fixed-film activated sludge (IFAS) process. Three pilot-scale startups were completed under the following conditions: anammox-only with preliminary biofilm carriers, PdNA with preliminary biofilm carriers, and PdNA with virgin carriers. AMX presence was confirmed via quantitative polymerase chain reaction (qPCR) after 57, 57, and 77 days, respectively. Prior to AMX detection, average influent concentrations of ammonia and nitrite ranged from 1.7-2.7 mg/L and 0.98-1.8 mg/L, respectively. This study demonstrated that AMX can be grown on carriers without AMX seeding under mainstream conditions (temperature 17-29°C, low ammonia and nitrite), regardless of whether nitrite came from upstream or partial denitrification within the reactor. This study also showed that using preliminary biofilm carriers can decrease startup time by approximately 1 month. These results address critical questions for moving mainstream anammox processes to full-scale implementation, and suggest that PdNA MBBRs are feasible and sustainable for full-scale ammonia, nitrate, and nitrite polishing to meet stringent total nitrogen requirements. PRACTITIONER POINTS: This research will help utilities develop methods for starting up mainstream anammox MBBRs without the barrier of anammox biomass seeding. Preliminary biofilm carriers accelerated startup time in a PdNA MBBR, but a virgin carrier reactor started up in a similar timeframe, contrary to expectations. Also, contrary to expectations, high concentrations of ammonia and nitrite are not necessary for startup of an anammox or PdNA MBBR.

Full-scale transition from denitrification to partial denitrification-anammox (PdNA) in deep-bed filters: Operational strategies for and benefits of PdNA implementation.

This study shows for the first time more than 2 years of operation of a mainstream anammox application at full-scale under temperate climate. This implementation of partial denitrification-anammox (PdNA) in deep bed filters at the HRSD York River treatment plant was demonstrated to achieve the benefits of shortcut nitrogen removal without nitrite oxidizing bacteria (NOB) out-selection. The transition from denitrification to PdNA filters required bleeding ammonium to the filters using an optimized ammonium versus NOx (AvN) control in the upstream aeration tanks and maintaining a nitrate residual in the filter effluent through feedforward/feedback control. The latter actions led to savings of 85% in methanol, 100% in alkalinity, and 35% in capacity enhancement. Up to 6 mg NH4 + -N/L with an average of 2.2 ± 0.98 mg NH4 + -N/L was removed through the anammox pathway, which accounted for about 15% of the overall plant nitrogen removal. Anammox enrichment was confirmed by activity testing and molecular analysis. The large excess of AnAOB capacity present in the filters (5-10 times more than normal operation) resulted in stable and reliable operation through winter conditions and showed potential for further intensification. PRACTITIONER POINTS: For the first time, long-term mainstream anammox was established full-scale through PdNA implementation in deep-bed filters. PdNA implementation required upstream aeration control optimization to provide a blend of ammonium and nitrate to the filters. Efficient anammox enrichment and retention resulted in reliable PdNA performance under different seasonal and influent conditions. PdNA implementation resulted in significant methanol and alkalinity savings and upstream capacity enhancement as ammonia removal depended less on aerobic nitrification. In the event of NOB out-selection and presence of nitrite, carbon savings in PdNA polishing filters can be enhanced via partial nitritation-anammox.

Comparison of sensor driven aeration control strategies for improved understanding of simultaneous nitrification/denitrification.

A pilot scale process was operated with A-stage effluent (ASE) and primary clarifier effluent (PCE) in MLE, all tanks aerated, A/O, and A2O configurations. Continuous DO control at high DO (2 mg/L), low DO (0.1-0.3 mg/L), ammonia-based aeration control (ABAC), and ammonia versus NOx (AvN) control (both continuous and intermittent operation) were compared on the basis of total inorganic nitrogen (TIN) removal, and simultaneous nitrification-denitrification (SND). The highly loaded adsorption/bio-oxidation (A/B) process configuration (4 hr HRT) with intermittent aeration was capable of achieving a maximum TIN removal of 80%, while the A2O process with PCE feed, an 11 hr HRT, and 0.2-0.3 mg/L DO continuous aeration achieved a maximum of 88% TIN removal. ABAC and AvN control did not always result in DO setpoints low enough to achieve SND, and even if setpoints were low enough to achieve SND that did not always result in increased overall TIN removal over continuous DO control of 2 mg/L. While there are other benefits to transitioning to sensor driven aeration control strategies such as ABAC and AvN, increased TIN removal during continuous aeration is not guaranteed. Results suggest that although low DO is a prerequisite for SND, carbon availability for denitrification in the aerobic zone is more likely to be the limiting factor once low DO conditions are met. PRACTITIONER POINTS: Intermittent aeration control results in higher TIN removal than continuous aeration at the same total SRT Continuous aeration AvN control is not likely to result in more TIN removal than continuous aeration ABAC for a given COD and nitrogen load Configurations that are designed to maximize predenitrification (e.g., MLE and A2O) are less likely to achieve increased SND in the aerobic zone from low DO operation than configurations that are not (e.g., A/O).