Candidatus Jettenia Research Articles

Successful start-up of anammox process from activated sludge and anaerobic sludge in a sequencing batch reactor using an unconventional strategy

In this study, the start-up of anammox in a sequencing batch reactor (SBR) using conventional activated and anaerobic sludge in 1:1 ratio (v/v) as inoculum was achieved using an unconventional strategy. The ratio of NH4+-N to NO2−–N in the influent was not maintained as per the stoichiometry (1:1.32) of anammox and rather varied depending on the effluent characteristics. Nitrite was omitted during its accumulation period to avoid its toxicity during the start-up phase. The SBR having polyurethane foam impregnated with activated carbon (Levapor® carriers) was operated for 212 d (temperature, 37 °C; influent pH, 8). Anammox process was started up in ~95 d with 85% average total nitrogen (TN) removal. Thereafter, the reactor showed average TN removal efficiency of 85% and exhibited an average NH4+-N and NO2−–N removals of 99.9% each at maximum influent NH4+-N and NO2−–N concentrations of 200 mg L−1 and 264 mg L−1, respectively. The 16S rRNA gene amplicon sequencing analysis of suspended sludge samples (100 d, 150 d, and 186 d) from the reactor revealed the dominance of infrequently occurring anammox bacterial genus Candidatus Jettenia in the reactor. The phyla Chloroflexi, Proteobacteria, and Bacteriodetes were also detected in the reactor.

Physiological stabilization, community characterization, and nitrogen degradation dynamics in an anammox consortium from estuarine sediments.

Anammox is a cost-effective and sustainable process for nitrogen removal; however, the production of a physiologically stable inoculum is a critical point in the start-up process. In this work, estuarine sediments were used as incubation seeds to obtain cultures with stable anammox activity. Assays were performed in batch cultures fed with stoichiometric amounts of ammonium and nitrite, analyzing physiological response variables and the microbial community. Estuarine sediments showed a stable anammox process after 90days, consuming ammonium and nitrite simultaneously with concomitant generation of N2 and nitrate in stoichiometric amounts. In kinetic assays, substrates were fully consumed after 210hr, exhibiting N2 and nitrate yields of 0.85 and 0.10, respectively. The microbial community analysis using PCR-DGGE indicated the presence of uncultured anammox bacteria and members of the genus Candidatus Jettenia. The results evidenced the achievement of anammox cultures, although their start-up and kinetic characteristics were less favorable than those recorded in man-made systems. PRACTITIONER POINTS: Estuarine sediments were used as incubation seeds to obtain cultures with stable anammox activity. The sediments were fed with stoichiometric amounts of ammonium and nitrite, analyzing the physiological response variables and the microbial community. Sediments showed a stable anammox process after 90days, converting the substrates into N2 and nitrate according to stoichiometry. Anammox cultures were achieved although their start-up and kinetic characteristics were less favorable than those recorded in man-made systems. Microbial community analysis using PCR-DGGE indicated the presence of uncultured anaerobic ammonia-oxidizing bacterium and members of genus Candidatus Jettenia.

Methanol tolerance and acclimation in the anammox process using a gel carrier

• Tolerant level of methanol was determined using MBBR using gel carriers. • No significant effect up to 30 mg L −1 , but irreversible inhibition at 40 mg L −1 . • By gradually increasing from 5 mg L −1 , no inhibitory effect even at 100 mg L −1 . • Heterotrophic denitrification contributed methanol consumption. • Methylotrophs such as Hyphomicrobiaceae was confirmed in the gel carriers. The methanol tolerance level of the anammox process was investigated in continuous feeding tests. In the conducted pulse methanol addition tests of gel carrier-immobilized anammox bacteria, the nitrogen removal performance was not significantly affected at influent methanol concentrations of up to 30 mg L −1 ; however, irreversible inhibition occurred at 40 mg L −1 . In the absence of methanol acclimation, the anammox process showed low methanol tolerance levels. Moreover, the stepwise addition of 5 mg L −1 of methanol resulted in no anammox inhibition even when the methanol concentration became 100 mg L −1 . The effluent total organic carbon concentration did not increase with the increase in the influent methanol concentration; this indicates that methanol was consumed along with the nitrate or nitrite by heterotrophic denitrification. These results show that the increase in the methanol tolerance level was due to the increase in the methanol consumption ability during the anammox process because of the increased denitrifying bacteria and not because the anammox bacteria exhibited resistance to methanol. The microbial community analysis revealed the dominant species in the gel carriers ( Candidatus Jettenia asiatica), existence of methylotrophic bacteria, and growth in the methanol-consuming bacterial population.

Effect of fulvic acid on bioreactor performance and on microbial populations within the anammox process

The long-term effect of fulvic acid (FA) on bioreactor performance and on microbial populations within the anammox process were firstly investigated in this study. The average nitrogen removal rate showed an upward trend when the influent TOC concentration of FA was 25.2–65.1 mg/L. However, when FA was increased to 80.3 mg/L, the reactor performance was slightly inhibited. In addition, judging from the particle size and settling properties, FA can promote anammox sludge granulation. After 53 days of exposure to FA, the genus Anaerolineaincreased in number, while Denitratisoma decreased. Candidatus Jettenia and Candidatus Kuenenia survived and enriched in the changed environment, potentially due to the interaction between anammox bacteria and some heterotrophic bacteria, which could protect anammox bacteria from adverse environments. These results indicate that FA can change the bacterial community and trigger different microbial interaction mechanisms within the anammox reactor.

Response and Adaptation of Microbial Community in a CANON Reactor Exposed to an Extreme Alkaline Shock

Responses of a microbial community in the completely autotrophic nitrogen removal over nitrite (CANON) process, which was shocked by a pH of 11.0 for 12 h, were investigated. During the recovery phase, the performance, anaerobic ammonia oxidation (anammox) activity, microbial community, and correlation of bacteria as well as the influencing factors were evaluated synchronously. The performance of the CANON process deteriorated rapidly with a nitrogen removal rate (NRR) of 0.13 kg·m−3·d−1, and Firmicutes, spore-forming bacteria, were the dominant phyla after alkaline shock. However, it could self-restore within 107 days after undergoing four stages, at which Planctomycetes became dominant with a relative abundance of 64.62%. Network analysis showed that anammox bacteria (Candidatus Jettenia, Kuenenia, and Brocadia) were positively related to some functional bacteria such as Nitrosomonas, SM1A02, and Calorithrix. Canonical correspondence analysis presented a strong correlation between the microbial community and influencing factors during the recovery phase. With the increase of nitrogen loading rate, the decrease of free nitrous acid and the synergistic effects, heme c content, specific anammox activity (SAA), NRR, and the abundance of dominant genus increased correspondingly. The increase of heme c content regulates the quorum sensing system, promotes the secretion of extracellular polymeric substances, and further improves SAA, NRR, and the relative abundance of the dominant genus. This study highlights some implications for the recovery of the CANON reactor after being exposed to an alkaline shock.

Metagenomic and metatranscriptomic insights into the complex nitrogen metabolic pathways in a single-stage bioreactor coupling partial denitrification with anammox

Combined partial-denitrification and anammox process has received growing interest for treating wastewaters with high nitrate concentrations, but the underlying molecular mechanisms are currently poorly understood due to the complexity in bacterial community composition and functionality of the coupled system. This study applied metagenomic and metatranscriptomic approaches to gain comprehensive and deep insights into the nitrogen metabolic pathways in a lab-scale partial-denitrification/anammox bioreactor showing stable nitrogen removal. Stoichiometric analysis showed that most of nitrogen was removed by the coupled system (~92.1%), but dissimilatory denitrification also contributed to nitrogen gas production. The latter was further evidenced by nosZ gene transcripts in the metatranscriptome. Metagenomic and metatranscriptomic analyses identified Candidatus Jettenia caeni as the most abundant and active anammox bacteria with relative abundance and mRNA expression of over 5% and 27%, respectively, while Thauera appeared as the major denitrifying bacteria in the system (relative abundance and mRNA expression exceeded 8% and 10%, respectively). mRNA levels of hdh/hzo and hzsA/B/C genes in Candidatus Jettenia caeni and Candidatus Brocadia sinica were much higher than those of other functional genes, confirming the major roles of the anammox bacteria in nitrogen metabolism. In addition, nrfA/B/C/D genes were mainly expressed in anammox bacteria, implying their involvement in that dissimilatory nitrite reduction to ammonia (especially for species Candidatus Brocadia sinica). Nitrate reduction process in Thauera was mainly achieved by the activation of the napA/B/C genes instead of narG/H genes. This study extablished the nitrogen metabolic network based on the comprehensive analyses of the abundance and mRNA expression of functional genes, which might improve our understanding of the nitrogen metabolic mechanisms of the combined partial-denitrification and anammox process.

Simultaneous anammox and denitrification process shifted from the anammox process in response to C/N ratios: Performance, sludge granulation, and microbial community

An up-flow anaerobic sludge blanket (UASB) reactor was operated to investigate the performance, sludge granulation, and microbial community dynamics of the simultaneous anammox and denitrification (SAD) process at different C/N ratios by inducing a shift from the anammox process. The nitrogen removal efficiency (NRE) increased with an increase in the C/N ratio at lower C/N ratios (≤ 0.5). The NRE and nitrogen removal rate (NRR) were 90.81% and 1.75 kgN m-3d-1, respectively, at a C/N ratio of 0.5. The contribution of the anammox process to nitrogen removal (Eanammox) decreased sharply to 51% from 81% as a result of the decreased activity of the anaerobic ammonia-oxidizing bacteria (AnAOB) at a C/N ratio of 0.9. The EPS production and the ratio of the protein fraction of EPS (EPSp) to the carbohydrate fraction of EPS (EPSc) increased with the increase in the C/N ratio (0.0-0.9), which induced sludge aggregation and resulted in larger particle size. The enrichment of denitrifying bacteria (DNB) on the surface of the AnAOB due to EPS bridging further enhanced sludge granulation. The Illumina sequencing results showed that the dominant genus of AnAOB (Candidatus Jettenia) initially increased with an increase in the C/N ratio at lower levels and then decreased sharply at a C/N ratio of 0.9. The relative abundance of Candidatus Jettenia was promoted by decreasing the C/N ratio, which supported the recovery of the AnAOB activity. Denitratisoma, Bacillus, Acinetobacter, and Pseudomonas were responsible for denitrification throughout the entire operational period.

Deciphering the genesis of anammox granular sludge floating from the perspective of microbial community

Sludge floating generally occurred in different anammox reactors, while the microbial difference between settling and floating sludge still unclear. In this study, the comprehensive investigation of microbial features between floating sludge and size-fractionated settling sludge was conducted in a stably operated anammox reactor. Firstly, scanning electron microscope image showed that mounts of long filament and a few gas tunnels were found in floating sludge, while cocci and bacilli were stacked in settling sludge. Microbial diversity was gradually decreased as the sludge aggregation, the microbial community structure and composition of floating sludge (FG) were more similar to large size-fractionated sludge (SG-L), and away from the small size-fractionated sludge (SG-S). Anammox bacteria Candidatus Jettenia tended to enrich in small size-fractionated sludge (accounting for 46.3%), while the large particle size feature and starved condition of floating sludge induced the decrease of Candidatus Jettenia (accounting for 23.5%). Meanwhile, denitrifying bacteria Dok59 rapidly enriched in large size-fractionated sludge and floating sludge, accounting for 8.9% and 9.8%, respectively. Network analysis showed that complex microbial interrelationships bring a robust microbial community in the SG-L sample, while the FG sample in the anammox reactor obtained an unstable microbial community. Moreover, the microbial community of FG has narrow niche breadths based on the low betweenness and node transitivity. These results obtained in this study extended our understanding of sludge floating in the anammox reactor and emphasized the crucial role of microorganisms.

Efficient transition from partial nitritation to partial nitritation/Anammox in a membrane bioreactor with activated sludge as the sole seed source

A lab-scale membrane bioreactor (MBR) was employed to carry out the partial nitritation/Anammox (PN/A) process from conventional activated sludge. Seed sludge was cultivated under microaerobic conditions for 10 days before seeding into the MBR. The bacterial community was analyzed on the basis of cloning and sequencing of 16S rRNA gene. Relative slow ammonia oxidation rates (3.2–13.0 mgN/L/d) were established in the microaerobic cultivation period. In the continuous MBR operation, the nitritation was achieved in the first 16 days and the reactor produced a balanced ratio between ammonia and nitrite which favored the proliferation of Anammox bacteria. Efficient transition from PN to PN/A was achieved in two months which was supported by appearance of reddish spots on the reactor inner wall and the concurrent consumption of ammonium and nitrite. The PN/A performed a robust and high-rate nitrogen removal capability and achieved a peak nitrogen removal of 1.81 kg N/m3/d. 16S rRNA gene-based analysis indicated that “Nitrosomonas sp.” and “Candidatus Jettenia sp.” accounted for ammonia oxidation and nitrogen depletion, respectively. Denitratisoma facilitated denitrification in the reactor. The present study suggested that a pre-cultivation of seed sludge under microaerobic conditions assists fast realization of PN and further convoyed efficient transition from PN to PN/A. Knowledge gleaned from this study is of significance to initiation, operation, and control of MBR-PN/As.

Construction and Denitrification Performance of A2/O Based on Partial Nitrification Coupled with an ANAMMOX System

To resolve the issue of existing municipal wastewater treatment plants (WWTPs) in China with an insufficient influent carbon source, a bench-scale A2/O process based on partial nitrification coupled with ANAMMOX was constructed by controlling aeration partition ratio, dissolved oxygen (DO) concentration, and sludge retention time (SRT). In this study, the nitrogen removal performance, nitrogen removal pathway, and microbial community structure of the system under different conditions were investigated. The results showed that the system had excellent nitrogen removal efficiency at low-C/N influent (C/N=5). The A2/O reactor had experienced the co-culture stage (Phase 1), screening stage (Phase 2-3), and enrichment stage (Phase 4) successively during the 140-day experiment, and the nitrogen removal pathway changed from nitrification and denitrification to partial nitrification coupled ANAMMOX in the end. The optimal removal efficiencies of 97.69% for NH4+-N and 87.83% for TN were obtained in the enrichment stage (Phase 4), and the effluent concentration of NH4+-N and TN were 1.20 mg·L-1 and 7.03 mg·L-1, respectively. Illumina MiSeq sequencing results showed that the enrichment of AOB including Nitrosomonas and Nitrosospira and the elimination of NOB including Nitrospira, Nitrococcus, and Nitrobacter were the main causes of achieving partial nitrification in the system. The enrichment of AnAOB including Candidatus Kuenenia and Candidatus Jettenia was the key point for the occurrence of ANAMMOX in the system, and thus, played an important role in the achievement of advanced nitrogen removal.

Feasibility of Adjusting the S2O32-/NO3- Ratio to Adapt to Dynamic Influents in Coupled Anammox and Denitrification Systems.

In this study, anammox, sulfur-based autotrophic denitrification, and heterotrophic denitrification (A/SAD/HD) were coupled in an expanded granular sludge bed (EGSB) reactor to explore the feasibility of enhancing denitrification performance by adjusting the S2O32−/NO3− (S/N) ratio to accommodate dynamic influents. The results indicated that the optimal influent conditions occurred when the conversion efficiency of ammonium (CEA) was 55%, the S/N ratio was 1.24, and the chemical oxygen demand (COD) was 50 mg/L, which resulted in a total nitrogen removal efficiency (NRE) of 95.0% ± 0.5%. The S/N ratio regulation strategy was feasible when the influent COD concentration was less than 100 mg/L and the CEA was between 57% and 63%. Characterization by 16S rRNA sequencing showed that Candidatus Jettenia might have contributed the most to anammox, while Thiobacillus and Denitratisoma were the dominant taxa related to denitrification. The findings of this study provide insights into the effects of CEA and COD on the performance of the A/SAD/HD system and the feasibility of the S/N ratio regulation strategy.

Treatment of low-strength ammonia wastewater by single-stage partial nitritation and anammox using upflow dual-bed gel-carrier reactor (UDGR)

This study investigated the single-stage partial nitritation and anammox (S-PNA) treatment of low-strength ammonia wastewater (≤140 mg NH4+-N/L). Upflow dual-bed gel-carrier reactor (UDGR) with polyvinyl alcohol cryogel biocarriers, developed in this study, was employed for the anammox biomass enrichment from conventional activated sludge and subsequent S-PNA experiments. Anammox biomass was successfully enriched from conventional activated sludge. The enriched anammox carriers were inoculated together with gel carriers containing nitrifying sludge into the S-PNA reactors. S-PNA activity developed rapidly, and the nitrogen removal efficiency and rate reached up to 90.1% (with complete ammonia removal) and 0.15 kg N/m3⋅d, respectively, under low nitrogen loading conditions (0.10–0.17 kg N/m3⋅d). The microbial community structure changed significantly while adapting to anammox and S-PNA conditions. Anammox was likely driven solely by a Candidatus Jettenia population accounting for ≤49.4% of bacterial 16S rRNA genes. The results demonstrate that the UDGR-based S-PNA is suitable for treating low-strength wastewater.

Full-scale simultaneous partial nitrification, anammox, and denitrification process for treating swine wastewater.

A full-scale swine-wastewater activated sludge treatment plant that contains naturally enriched anammox biofilms was investigated for 2 years. Red biofilm in this system included Planctomycetes at a maximum of 62.5% of the total bacteria diversity, including Candidatus Jettenia and Candidatus Brocadia. The plant was operated with an influent containing 1,104 ± 513 mg/L biochemical oxygen demand (BOD) and 629 ± 198 mg/L total nitrogen (TN) (BOD/N of 1.78 ± 0.58) at a volumetric BOD loading rate of 0.32 ± 0.12 kg/m3/d. Notwithstanding drastically varying influent concentrations, BOD removal efficiency was stable at 95 ± 4%. However, TN removal fluctuated at 75 ± 14%. Dissolved oxygen (DO) concentrations in the aeration tank were 0.06-2.0 mg/L. DO concentration greatly affected nitrogen removal, e.g. when DO was lower than 0.3 mg/L, total inorganic nitrogen removal was 61 ± 14% (≤20 °C), 78 ± 16% (20-30 °C), and 75 ± 12% (≥30 °C), whereas at higher DO concentrations, removal rates were 47 ± 13%, 55 ± 16%, and 68%, respectively. As BOD concentration in the influent was limited compared to nitrogen concentration, nitrogen was likely removed by simultaneous nitrification, anammox, and denitrification (SNAD) under microaerobic conditions. Maintaining low DO concentrations would therefore be a simple method to improve nitrogen removal during SNAD processes for swine-wastewater treatment with fluctuating influent.

Shift in bacterial community structure in response to salinity in a continuous anaerobic ammonium oxidation (anammox) reactor

The effect of gradual salt adaptation on nitrogen removal performance was examined in a continuous flow anaerobic ammonium oxidation (anammox) process. Specific anammox activity (SAA) batch test was conducted to observe the effect of salinity shock loads. SAA rapidly reduced from 100% to 2.3% when salinity increased from 0 g.L−1 to 5.0 g.L−1 NaCl. On the contrary, high total nitrogen removal efficiency (TNRE) of 85% was obtained when salt concentrations gradually increased up to 25.0 g.L−1 NaCl, after 264 d of the continuous anammox operation. Then, the additional increase of salinity by 2.5 g.L−1 resulted in critical negative effect of 63.8% reduction in TNRE. Candidatus Jettenia sp. was dominant as a salt-tolerant anammox species in the salt ranges of 0–27.5 g.L−1 NaCl. In contrast, Candidatus Kuenenia stuttgartiensis was observed at 10–27.5 g.L−1 NaCl with smaller relative abundance than that of Ca. Jettenia sp. This indicates that Ca. Kuenenia sp. would build limited niche in the competition with Ca. Jettenia sp. under sudden increase of salinity stress. Interestingly, heterotrophic bacteria became dominant in autotrophic nitrogen removal process in response to the increase in salt concentration, which could be a cause of anammox system failure.

Enhanced selective enrichment of partial nitritation and anammox bacteria in a novel two-stage continuous flow system using flat-type poly (vinylalcohol) cryogel films

To improve stability of nitrogen removal in partial nitritation (PN)-anammox process, flat-type cryogel films using poly (vinylalcohol) named as FT-CPVAF were applied in continuous reactors. Stable PN operation was maintained with short acclimation of 8 days and ammonium oxidation rate of 1.68 ± 0.12 kg N m−3 d−1 comparatively higher than previous studies. The nitrogen removal, initially inhibited by an oxygen shock, was immediately reactivated with short lag-period by immobilization of anammox bacteria in FT-CPVAF. A novel two-stage PN-anammox process was operated in a continuous flow using FT-CPVAF for treatment of ammonium-rich synthetic wastewater (influent 315 mg NH4+-N L−1) showing 89.6 ± 0.76% of nitrogen removal at short hydraulic retention time (7.7 h). The use of FT-CPVAF enhanced selective enrichment of AOB and anammox bacter ia confirmed by high-throughput sequencing of i.e., relative abundances of Nitrosomonas europaea C-31 (37.14% in PN reactor) and ‘Candidatus Jettenia caeni’ (34.36% in anammox reactor).

Genome of a Novel Bacterium "Candidatus Jettenia ecosi" Reconstructed From the Metagenome of an Anammox Bioreactor.

The microbial community of a laboratory-scale bioreactor based on the anammox process was investigated by using metagenomic approaches and fluorescent in situ hybridization (FISH). The bioreactor was initially inoculated with activated sludge from the denitrifying bioreactor of a municipal wastewater treatment station. By constantly increasing the ammonium and nitrite load, a microbial community containing the novel species of anammox bacteria “Candidatus Jettenia ecosi” developed in the bioreactor after 5 years when the maximal daily nitrogen removal rate reached 8.5 g/L. Sequencing of the metagenome of anammox granules and the binning of the contigs obtained, allowed a high quality draft genome of the dominant anammox bacterium, “Candidatus Jettenia ecosi” to be assembled. Annotation of the 3.9 Mbp long genome revealed 3970 putative protein-coding genes, 45 tRNA genes, and genes for 16S/23S rRNAs. Analysis of the genome of “Candidatus Jettenia ecosi” revealed genes involved in anammox metabolism, including nitrite and ammonium transporters, copper-containing nitrite reductase, a nitrate reductase complex, hydrazine synthase, and hydrazine dehydrogenase. Autotrophic carbon fixation could be accomplished through the Wood Ljungdahl pathway. The composition of the community was investigated through a search of 16S rRNA sequences in the metagenome and FISH analysis of the anammox granules. The presence of the members of Ignavibacteriae, Betaproteobacteria, Chloroflexi and other microbial lineages reflected the complexity of the microbial processes in the studied bioreactor performed by anammox Planctomycetes, fermentative bacteria, and denitrifiers.

Anaerobic ammonium oxidation is a major N-sink in aquifer systems around the world.

Global-scale N-oxide contamination of groundwater within aquifers occurs due to the widespread use of N-bearing fertilizers and chemicals, threatening both human and environmental health. However, the conversion of these pollutants in active nitrogen (N) cycling processes in the subsurface biosphere still remains unclear. This study investigates the global occurrence of anaerobic ammonium oxidation (anammox) in aquifers, where anammox was found to be turned on and off between saturated and unsaturated soil horizons, and contributed 36.8-79.5% to N loss in saturated soil horizons, the remainder being due to denitrification which has traditionally been considered the main pathway for removal of N-pollutants from aquifers. Although anammox activity was undetectable in the unsaturated soil horizons, it could potentially be activated by contact with ascending groundwater. High-throughput pyrosequencing analysis identified Candidatus Brocadia anammoxidans as being the most abundant anammox bacterium in the saturated soils investigated. However, the anammox bacterial abundance was determined by the relative richness of Candidatus Jettenia asiatica. Isotopic pairing experiments revealed that coupling anammox with ammonium oxidation and respiratory ammonification enabled the formation of a revised N cycle in aquifer systems, in which respiratory ammonification acted as an important coordinator. Anammox can therefore contribute substantially to aquifer N cycling and its role in remediation of aquifers contaminated with N-oxides may be of global importance.

Nitrogen removal from food waste digestate using partial nitritation-anammox process: Effect of different aeration strategies on performance and microbial community dynamics

The feasibility of employing anammox and partial nitritation-anammox (PN/A) processes for nitrogen removal from food waste (FW) digestate was investigated in this study. The effects of different aeration strategies on the microbial community were also investigated. To achieve this, after anammox enrichment (Phase 1), the reactor was fed with digestate supplemented with nitrite (Phase 2), and subsequently different aeration strategies were evaluated to establish PN/A. Aeration strategies with high anoxic periods (30 and 45 min) in relation to aerobic periods (15 min) coupled with low air flow rates (0.026 L min−1. Lreator−1) were found to be better for establishing PN/A, as coefficients of produced nitrate/removed ammonium were closer to those reported previously (0.17 and 0.21). Aeration conditions considerably altered the microbial community. Candidatus Brocadia was replaced by Candidatus Jettenia, after the first aeration strategies. These results support the feasibility of FW digestate treatment using anammox and PN/A processes and provide a better understanding of the effect of aeration on microbial dynamics in PN/A reactors.

Genome-centered omics insight into the competition and niche differentiation of Ca. Jettenia and Ca. Brocadia affiliated to anammox bacteria.

Although the niche differentiation of anammox bacteria has been extensively observed in a lab-scale reactor, the inherent mechanism for this ecological phenomenon is still elusive. Here, we combined the long-term reactor operation, genome-centered metagenome, and metatranscriptome analyses to gain insight into the substrate competition and niche differentiation of Candidatus Jettenia and Candidatus Brocadia. After 146 days of operation, we found the anammox bacterial population shifted from Ca. Jettenia to Ca. Brocadia in the immobilization-anaerobic baffled reactor (I-ABR) with the ammonium and nitrite concentrations of 30 mg/L. Importantly, the genome and transcript comparisons of Ca. Jettenia and Ca. Brocadia showed that Ca. Brocadia harbored more complete function in cell chemotaxis, flagellar assembly, and two-component system and more redundant function in nitrite reduction, in which the genes were also highly expressed. Ca. Brocadia out-competed Ca. Jettenia at the mainstream condition. Meanwhile, though the highest biomass concentration led to the highest nitrogen removal rate (NRR) in the first compartment (C1), the competition of Ca. Jettenia and Ca. Brocadia could also affect the NRR of different compartments through affecting the bacterial activity. Substrate competition of anammox bacteria led to higher transcript activity of Ca. Jettenia and Ca. Brocadia in the second (C2) and fourth (C4) compartments, respectively. Further, high transcript activity of Ca. Brocadia led to the higher NRR in C4. A comparison of metabolic potential based on the metagenome-assembled genome adds a different dimension for understanding the discrepantly physiological characteristics and competition of anammox bacteria for wastewater treatment.

Discrepant gene functional potential and cross-feedings of anammox bacteria Ca. Jettenia caeni and Ca. Brocadia sinica in response to acetate

Although the enhancement of anammox performance for wastewater treatment due to the addition of small amount of acetate has been reported, discrepant metabolic responses of different anammox species have not been experimentally evaluated. Based on metagenomics and metatranscriptomic data, we investigated the competitiveness between two typical anammox species, Candidatus Jettenia caeni (J. caeni) and Candidatus Brocadia sinica (B. sinica), in anammox consortia under mixotrophic condition, where complex metabolic interactions among anammox bacteria and heterotrophs also changed with acetate addition. Contrary to J. caeni, the dissimilatory nitrate reduction to ammonium pathway of B. sinica was markedly stimulated for improving nitrogen removal. More acetate metabolic pathways and up-regulated AMP-acs expression for acetyl-CoA synthesis in B. sinica contributed to its superiority in acetate utilization. Interestingly, cross-feedings, including the nitrogen cycle, amino acid cross-feeding and B-vitamin metabolic exchange between B. sinica and other heterotrophs seemed to be enhanced with acetate addition, contributing to a reduction in metabolic energy cost to the whole community. Our work not only clarified the mechanism underlying discrepant responses of different anammox species to acetate, but also suggests a possible strategy for obtaining higher nitrogen removal rates in wastewater treatment under low C/N ratio.