Anammox Activity Research Articles

Enhancing nitrogen removal of carbon-limited municipal wastewater in step-feed biofilm batch reactor through integration of anammox

The biological nitrogen removal of municipal wastewater was successfully improved by integrating anammox in a step-feed sequencing biofilm batch reactor. Despite fluctuating influent carbon to nitrogen ratio (1.9–5.1) and decreasing temperature (24.1–16.3 ℃), nitrogen removal efficiency of 95.9 ± 1.4 % and nitrogen removal rate of 0.23 ± 0.02 kg N/(m3·d) were successfully maintained without requirement of external carbon sources. The advanced removal performance was mainly attributed to the enhanced anammox. Anammox bacteria presented a high relative abundance (42.9% in biofilms, 1.5% in flocs) and anammox activity was as high as 5.42 ± 0.12 mg N/(g volatile suspended solids·h). Further analysis suggested that flexible control of influent organic and ammonium through step-feeding could provide multiple substrate supply for anammox reaction, potentially resulting in stable combination of anammox with hybrid-nitrite-shunt processes. Overall, this study provides a promising anammox-related application with simple-control step-feed strategy for enhanced and stable nitrogen removal from carbon-limited municipal wastewater.

Complete nitrogen removal from low-strength wastewater via double nitrite shunt coupling anammox and endogenous nitrate respiration: Functional metabolism and electron transport

Autotrophic nitrogen removal processes based on anammox are facing huge challenges of high effluent quality in wastewater treatment. In this study, a novel hybrid system of anammox coupled with heterotrophic endogenous metabolisms was developed in a single-stage bioreactor to treat real domestic wastewater. Complete nitrogen removal was achieved at a low carbon to nitrogen (C/N) ratio of 2.8 and more than 95.2% nitrogen removal was maintained under an extremely low C/N ratio of 1.9. The nitrite shunt supplied by dual partial pathways (partial nitritation and endogenous partial denitratation) guaranteed the anammox activity with a high contribution to nitrogen removal above 60%. While endogenous denitrification (ED) and denitrifying phosphorus removal (DPR) both participated in nitrate respiration, contributing more than 28% and 9% to nitrogen removal, respectively. The coexistence and enrichment of anammox bacteria (Ca. Brocadia, 0.9%), glycogen accumulating organisms (Ca. Competibacter, 6.4%) and phosphorus accumulating organisms (Dechloromonas, 3.9% and Ca. Accumulibacter, 0.7%) contributed to the co-metabolism of carbon, nitrogen and phosphorus. The functional genes encoding the above metabolism were highly correlated with the dominant strains, indicating that the multiple pathways of carbon and nitrogen metabolism supported the continuous supply of electrons required for nitrogen removal. This study puts forward a novel complete nitrogen removal process by successfully coupling autotrophic and endogenous heterotrophic metabolism.

Fast start-up of anammox process: Effects of extracellular polymeric substances addition on performance, granule properties, and bacterial community structure

The slow startup is the major obstacle to the application of anaerobic ammonium oxidation (anammox) process in mainstream wastewater treatment. Extracellular polymeric substances (EPS) are one potential resource for stable anammox reactor operation. Response surface analysis was used to optimize the specific anammox activity (SAA) with the addition of EPS; SAA was maximum at a temperature of 35 °C and the EPS concentration of 4 mg/L. By comparing the nitrogen removal of anammox reactors with no EPS (R0), immobilized EPS (EPS-alginate beads) (R1), and liquid EPS (R2), we found that EPS-alginate beads significantly speed up the startup of anammox process and enable the start time to be shortened from 31 to 19 days. As a result of the higher MLVSS content, higher zeta potential, and lower SVI30, anammox granules of R1 exhibited a stronger capacity to aggregate. Moreover, EPS extracted from R1 had higher flocculation efficiencies than EPS derived from R0 and R2. Phylogenetic analysis of 16S rRNA genes revealed that the main anammox species in R1 is Kuenenia taxon. To clarify the relative significance of stochastic vs deterministic processes in the anammox community, neutral model and network analysis are employed. In R1, community assembly became more deterministic and stable than in other cultures. Our results show that EPS might inhibit heterotrophic denitrification and thereby promote anammox activity. This study suggested a quick start-up strategy for the anammox process based on resource recovery, which is helpful for environmentally sustainable and energy-efficient wastewater treatment.

Efficient nitrogen removal in a total floc sludge system from domestic wastewater with low C/N: High anammox nitrogen removal contribution driven by endogenous partial denitrification

Since unsustainable partial nitrification prone to unstable nitrogen removal rates, cultivation and enrichment of AnAOB, further improve autotrophic nitrogen removal contribution have been challenges in the mainstream anammox process. This study proposed a new strategy to enrich AnAOB motivated by endogenous partial denitrification (EPD) in total floc sludge system through the AOA process with sustainable nitrification. The results showed that in the presence of NH4+ and NO3– at the anoxic stage of N-EPDA, Ca. Brocadia was enriched (0.005%→0.92%) in floc sludge via internal carbon source metabolism of EPD. The C/N and temperature of N-EPDA were also optimized to achieve higher activities of EPD and anammox. The N-EPDA was operated at low C/N ratio (3.1) with anammox nitrogen removal contribution of 78% during the anoxic stage, Eff.TIN of 8.3 mg/L and NRE of 83.5% during phase III, achieved efficient autotrophic nitrogen removal and AnAOB enrichment in the absence of partial nitrification.

Response of granular anammox process under mainstream conditions to continuous and transient organic loads

Even small fluctuations in the amount of organic matter in wastewater significantly affect the structure and function of anammox microbial communities under mainstream conditions with low ammonium concentrations, because a high organic C/N ratio causes heterotrophic denitrifying bacteria to outcompete anammox bacteria. This paper presents a comprehensive investigation of the effects of different levels of continuous and transient organic loads on nitrogen removal and sludge characteristics in upflow granular anammox reactors under mainstream conditions. Continuous organic load at 30 mg chemical oxygen demand (COD)/L influent improved nitrogen removal efficiency without compromising anammox performance. However, exposure to higher levels of continuous organic load (≥60 mg COD/L) caused significant inhibition of anammox activity (complete inhibition at 150 mg COD/L). The performance degradation was accompanied by the destabilization of anammox granular sludge, with a decrease in tightly bound protein and increase in loosely bound polysaccharide contents in the sludge extracellular polymeric substances. Further, DNA sequencing analysis showed that heterotrophic denitrifying Rhodocyclaceae and Pseudomonadaceae bacteria collectively accounted for 36.8–61.3% of the total bacterial reads. These bacteria emerged and outcompeted anammox Ca. Brocadiaceae (accounting for less than 8.4% of the total reads) with increasing organic load, resulting in a significant decrease in anammox activity. Meanwhile, the inhibitory effect of transient organic load (one-day shock loading) was fully reversed within one day at both 150 and 300 mg COD/L shock levels, although the latter caused a sudden fall in anammox performance. The overall results suggest that loading an appropriately low amount of organic matter can provide a means to improve the nitrogen removal performance of anammox granular sludge without sacrificing anammox activity and granule stability under mainstream conditions.

Biochar accelerates the start-up of the anammox process: Phenomenon and potential mechanisms

Biochar was applied to accelerate the establishment of the anaerobic ammonium oxidation (anammox) reactor without temperature control (10 to 37 °C). 5 g/L coconut and apricot charcoal were added in R1 and R2. As a result, it took 121, 85 and 90 days for the start-up of R0, R1 and R2. The anammox pathway related functional gene (hzsB) in the bottom region of R0, R1 and R2 were 4.86 × 1011, 1.31 × 1012 and 5.85 × 1011 copies/g after 215-day operation which was significantly greater than that in the seeding sludge (2.56 × 107 copies/g). Candidatus Kuenenia and C. Brocadia were enriched in all reactors and the maximum nitrogen removal rate of R0, R1 and R1 were 1.29, 1.41 and 1.48 kgN/(m3·d), respectively. Biochar promoted the electron transfer capacity, resulting in the increase of functional gene responsible for the anammox pathway. The anammox microbes were enriched and specific anammox activity and nitrogen removal efficiency were improved eventually. The findings in our study could provide an alternative to establish the anammox process rapidly without temperature control.

Double-edged sword effects of dissimilatory nitrate reduction to ammonium (DNRA) bacteria on anammox bacteria performance in an MBR reactor

Dissimilatory nitrate reduction to ammonium (DNRA) bacteria imposing double-edged sword effects on anammox bacteria were investigated in an anammox-membrane bioreactor (MBR) experiencing an induced crash-recovery event. During the experiment, the anammox-MBR was loaded with NH4+-N:NO2−-N ratios (RatioNH4+-N: NO2−-N) of 1.20–1.60. Initially, the anammox-MBR removed over 95% of 100 mg/L NH4+-N and 132 mg/L NO2−-N (RatioNH4+-N: NO2−-N = 0.76, the well accepted stoichiometric RatioNH4+-N: NO2−-N for anammox) in the influent (Stage 0). Then, we induced a system crash-recovery event via nitrite shock loadings to better understand responses from different guilds of bacteria in anammox-MBR, loaded with 1.60 RatioNH4+-N: NO2−-N with 100 mg/L NO2−-N in the influent (Stage 1). Interestingly, the nitrogen removal by anammox bacteria was maintained for about 20 days before starting to decrease significantly. In Stage 2, we further increased influent nitrite concentration to 120 mg/L (1.33 RatioNH4+-N: NO2−-N) to simulate a high nitrite toxicity scenario for a short period of time. As expected, nitrogen removal efficiency dropped to only 16.8%. After the induced system crash, anammox-MBR performance recovered steadily to 93.2% nitrogen removal with a 1.25 RatioNH4+-N:NO2−-N and a low nitrite influent concentration of 80 mg/L NO2−-N. Metagenomics analysis revealed that a probable causality of the decreasing nitrogen removal efficiency in Stage 1 was the overgrowth of DNRA-capable bacteria. The results showed that the members within the Ignavibacteriales order (21.7%) out competed anammox bacteria (17.0%) in the anammox-MBR with elevated nitrite concentrations in the effluent. High NO2−-N loading (120 mg N/L) further caused the predominant Candidatus Kuenenia spp. were replaced by Candidatus Brocadia spp. Therefore, it was evident that DNRA bacteria posed negative effects on anammox with 1.60 RatioNH4+-N: NO2−-N. Also, when 120 mg/L NO2−-N fed to anammox-MBR (RatioNH4+-N: NO2−-N = 1.33), canonical denitrification became the primary nitrogen sink with both DNRA and anammox activities decreased. They probably fed on lysed microbial cells of anammox and DNRA. In Stage 3, a low RatioNH4+-N: NO2−-N (1.25) with 80 mg/L NO2−-N was used to rescue the system, which effectively promoted DNRA-capable bacteria growth. Although anammox bacteria's abundance was only 7.7% during this stage, they could be responsible for about 90% of the total nitrogen removal during this stage.

Integration of EBPR with mainstream anammox process to treat real municipal wastewater: Process performance and microbiology

The mainstream application of anaerobic ammonium oxidation (anammox) for sustainable N removal remains a challenge. Similarly, with recent additional stringent regulations for P discharges, it is imperative to integrate N with P removal. This research studied integrated fixed film activated sludge (IFAS) technology to simultaneously remove N and P in real municipal wastewater by combining biofilm anammox with flocculent activated sludge for enhanced biological P removal (EBPR). This technology was assessed in a sequencing batch reactor (SBR) operated as a conventional A2O (anaerobic-anoxic-oxic) process with a hydraulic retention time of 8.8 h. After a steady state operation was reached, robust reactor performance was obtained with average TIN and P removal efficiencies of 91.3 ± 4.1% and 98.4 ± 2.4%, respectively. The average TIN removal rate recorded over the last 100 d of reactor operation was 118 mg/L·d, which is a reasonable number for mainstream applications. The activity of denitrifying polyphosphate accumulating organisms (DPAOs) accounted for nearly 15.9% of P-uptake during the anoxic phase. DPAOs and canonical denitrifiers removed approximately 5.9 mg TIN/L in the anoxic phase. Batch activity assays, which showed that nearly 44.5% of TIN were removed by the biofilms during the aerobic phase. The functional gene expression data also confirmed anammox activities. The IFAS configuration of the SBR allowed operation at a low solid retention time (SRT) of 5-d without washing out biofilm ammonium-oxidizing and anammox bacteria. The low SRT, combined with low dissolved oxygen and intermittent aeration, provided a selective pressure to washout nitrite-oxidizing bacteria and glycogen-accumulating organisms, as relative abundances of

Evaluating the suitability of granular anammox biomass for nitrogen removal from vegetable tannery wastewater

In the present study, the potential inhibitory effect of biologically pre-treated vegetable tannery wastewater (TW) on anammox granular biomass was evaluated. Beside high organic and chemicals load, vegetable TW are characterised by high salinity and high tannins concentration, the latter belonging to a group of bio-refractory organic compounds, potentially inhibitory for several bacterial species. Recalcitrant tannin-related organic matters and salinity were selected as the two potential inhibitory factors and studied either for their separate and combined effect. Parallel batch tests were performed, with biomass acclimated and non-acclimated to salinity, testing three different conditions: non-saline control test with non-acclimated biomass (CT); saline control test with acclimated biomass (SCT); vegetable tannery wastewater test with acclimated biomass (TWT). Compared with non-saline CT, the specific anammox activity in tests SCT and TWT showed a reduction of 28 and 14%, respectively, suggesting that salinity, at conductivity values of 10 mS/cm (at 25 °C), was the main impacting parameter. As a general conclusion, the study reveals that there is no technical limitation for the application of the anammox process to vegetable TW, but preliminary biomass acclimation as well as regular biomass activity monitoring is recommended in case of long-term applications. To the best of our knowledge, this is the first work assessing the impact of vegetable TW on anammox biomass.

A novel strategy for a reinforced anammox process with iron-modified Enteromorpha prolifera biochar

Modified biochar with higher electron transport and adsorption capabilities could significantly improve the performance of anaerobic ammonia oxidation (anammox). However, there are few related investigations on the reinforcement of anammox through iron-modified Enteromorpha prolifera biochar (IMEPB). In this study, with the addition of the IMEPB in the anammox system, the enhancing process of anammox performance was studied, the improving feasibility of anammox was evaluated, and the reinforcing mechanism of anammox was elucidated. The results showed that the optimal iron−charcoal ratio (Fe:C) and IMEPB dosage were 1:10 and 10 g L−1, respectively. Under the optimal conditions, when the nitrogen loading rate gradually increased to 0.557 (kg m−3 day−1), the nitrogen removal efficiency and nitrogen removal rate of the anammox process supplemented with IMEPB increased by 11%, and the specific anammox activity increased by 23.8%. Compared with the control, the secretion of extracellular polymeric substances (EPS) of anammox bacteria supplemented IMEPB increased by 24.4%, greatly improving the stability of the anammox system. Meanwhile, EPS secretion further promoted the microbial activity of anammox bacteria, achieving a 19% increase in the abundance of Candidatus Brocadia. These findings demonstrate the potential mechanism of IMEPB in improving anammox, provide new insights into recycling E. prolifera, and provide a novel reinforcement strategy for anammox. In the future, adding IMEPB may be a vital measure for the practical application of anammox in coastal areas.Graphical

Effects of reducing, stabilizing, and antibiotic agents on “Candidatus Kuenenia stuttgartiensis”

Anaerobic ammon ium oxidizing (anammox) bacteria oxidize ammonium and reduce nitrite, producing N2, and could play a major role in energy-optimized wastewater treatment. However, sensitivity to various environmental conditions and slow growth currently hinder their wide application. Here, we attempted to determine online the effect of environmental stresses on anammox bacteria by using an overnight batch activity test with whole cells, in which anammox activity was calculated by quantifying N2 production via headspace-pressure monitoring. A planktonic mixed culture dominated by “Candidatus Kuenenia stuttgartiensis” strain CSTR1 was cultivated in a 30-L semi-continuous stirring tank reactor. In overnight resting-cell anammox activity tests, oxygen caused strong inhibition of anammox activity, which was reversed by sodium sulfite (30 µM). The tested antibiotics sulfamethoxazole, kanamycin, and ciprofloxacin elicited their effect on a dose-dependent manner; however, strain CSTR1 was highly resistant to sulfamethoxazole. Anammox activity was improved by activated carbon and Fe2O3. Protein expression analysis from resting cells after anammox activity stimulation revealed that NapC/NirT family cytochrome c (KsCSTR_12840), hydrazine synthase, hydrazine dehydrogenase, hydroxylamine oxidase, and nitrate:nitrite oxidoreductase were upregulated, while a putative hydroxylamine oxidoreductase HAO (KsCSTR_49490) was downregulated. These findings contribute to the growing knowledge on anammox bacteria physiology, eventually leading to the control of anammox bacteria growth and activity in real-world application.Key Points• Sulfite additions can reverse oxygen inhibition of the anammox process• Anammox activity was improved by activated carbon and ferric oxide• Sulfamethoxazole marginally affected anammox activityGraphical abstract

Rapid Start-Up Characteristics of Anammox under Different Inoculation Conditions

The long multiplication time and extremely demanding enrichment environment requirements of Anammox bacteria (AAOB) have led to difficult reactor start-ups and hindered its practical dissemination. Few feasibility studies have been reported on the recovery of AAOB activity initiation after inlet substrate disconnection caused by an unfavorable condition, and few factors, such as indicators of the recovery process, have been explored. Therefore, in this experiment, two modified expanded granular sludge bed reactors (EGSB) were inoculated with 1.5 L anaerobic granular sludge (AGS) + 1 L Anammox sludge (AMS) (R1) and 2.5 L anaerobic granular sludge (AGS) (R2), respectively. After a long-term (140 days) starvation shock at a high temperature (38 °C), the bacteria population activity recovery experiments were conducted. After 160 days, both reactors were successfully started up, and the total nitrogen removal rates exceeded 87%. Due to the experimental period, the total nitrogen removal rate of R2 was slightly higher than that of R1 in the final stage. However, it is undeniable that R2 had a relatively long activity delay during startup, while R1 had no significant activity delay during startup. The sludge obtained from R1 had a higher specific anammox activity (SAA). Analysis of the extracellular polymer substances (EPS) results showed that the extracellular polymer content in R1 was higher than that in R2 throughout the recovery process, indicating that R1 had higher sludge stability and denitrification performance. Scanning electron microscopy (SEM) analysis showed that more extracellular filamentous bacteria could be seen in the R1 reactor with better morphology of Anammox bacteria. In contrast, the R2 reactor had fewer extracellular hyphae and micropores as a percentage and higher filamentous bacteria content. The results of microbial 16SrDNA analysis showed that R1 used AAOB as inoculum to initiate Anammox, and the reactor was enriched with Anammox bacteria earlier and in much greater abundance than R2. The experimental results indicated that inoculating mixed anaerobic granular sludge and Anammox sludge to initiate an anammox reactor was more effective.

Feasibility of packed-bed trickling filters for partial nitritation/anammox: Effects of carrier material, bottom ventilation openings, hydraulic loading rate and free ammonia

This study pioneers the feasibility of cost-effective partial nitritation/anammox (PN/A) in packed-bed trickling filters (TFs). Three parallel TFs tested different carrier materials, the presence or absence of bottom ventilation openings, hydraulic loading rates (HLR, 0.4–2.2 m3 m−2 h−1), and free ammonia (FA) levels on synthetic medium. The inexpensive Argex expanded clay was recommended due to the similar nitrogen removal rates as commercially used plastics. Top-only ventilation at an optimum HLR of 1.8 m3 m−2 h−1 could remove approximately 60% of the total nitrogen load (i.e., 300 mg N L-1 d−1, 30 °C) and achieve relatively low NO3–-N accumulation (13%). Likely FA levels of around 1.3–3.2 mg N L-1 suppressed nitratation. Most of the total nitrogen removal took place in the upper third of the reactor, where anammox activity was highest. Provided further optimizations, the results demonstrated TFs are suitable for low-energy shortcut nitrogen removal.

Coupling of Anammox Activity and PAH Biodegradation: Current Insights and Future Directions

Anaerobic ammonium oxidation (anammox) has shown success in past years for the treatment of municipal and industrial wastewater containing inorganic nutrients (i.e., nitrogen). However, the increase in polycyclic aromatic hydrocarbon (PAH)-contaminated matrices calls for new strategies for efficient and environmentally sustainable remediation. Therefore, the present review examined the literature on the connection between the anammox process and PAHs using VOSviewer to shed light on the mechanisms involved during PAH biodegradation and the key factors affecting anammox bacteria. The scientific literature thoroughly discussed here shows that PAHs can be involved in nitrogen removal by acting as electron donors, and their presence does not adversely affect the anammox bacteria. Anammox activity can be improved by regulating the operating parameters (e.g., organic load, dissolved oxygen, carbon-to-nitrogen ratio) and external supplementation (i.e., calcium nitrate) that promote changes in the microbial community (e.g., Candidatus Jettenia), favoring PAH degradation. The onset of a synergistic dissimilatory nitrate reduction to ammonium and partial denitrification can be beneficial for PAH and nitrogen removal.

Characterization of Enrichment Cultures of Anammox, Nitrifying and Denitrifying Bacteria Obtained from a Cold, Heavily Nitrogen-Polluted Aquifer

Anammox bacteria related to Candidatus Scalindua were recently discovered in a cold (7.5 °C) aquifer near sludge repositories containing solid wastes of uranium and processed polymetallic concentrate. Groundwater has a very high level of nitrate and ammonia pollution (up to 10 and 0.5 g/L, respectively) and a very low content of organic carbon (2.5 mg/L). To assess the potential for bioremediation of polluted groundwater in situ, enrichment cultures of anammox, nitrifying, and denitrifying bacteria were obtained and analyzed. Fed-batch enrichment of anammox bacteria was not successful. Stable removal of ammonium and nitrite (up to 100%) was achieved in a continuous-flow reactor packed with a nonwoven fabric at 15 °C, and enrichment in anammox bacteria was confirmed by FISH and qPCR assays. The relatively low total N removal efficiency (up to 55%) was due to nonstoichiometric nitrate buildup. This phenomenon can be explained by a shift in the metabolism of anammox bacteria towards the production of more nitrates and less N2 at low temperatures compared to the canonical stoichiometry. In addition, the too high an estimate of specific anammox activity suggests that N cycle microbial groups other than anammox bacteria may have contributed significantly to N removal. Stable nitrite production was observed in the denitrifying enrichment culture, while no "conventional" nitrifiers were found in the corresponding enrichment cultures. Xanthomonadaceae was a common taxon for all microbial communities, indicating its exclusive role in this ecosystem. This study opens up new knowledge about the metabolic capabilities of N cycle bacteria and potential approaches for sustainable bioremediation of heavily N-polluted cold ecosystems.

Comparative assessment of energy generation from ammonia oxidation by different functional bacterial communities

Bioelectrochemical ammonia oxidation (BEAO) in a microbial fuel cell (MFC) is a recently discovered process that has the potential to reduce energy consumption in wastewater treatment. However, level of energy and limiting factors of this process in different microbial groups are not fully understood. This study comparatively investigated the BEAO in wastewater treatment by MFCs enriched with different functional groups of bacteria (confirmed by 16S rRNA gene sequencing): electroactive bacteria (EAB), ammonia oxidizing bacteria (AOB), and anammox bacteria (AnAOB). Ammonia oxidation rates of 0.066, 0.083 and 0.082 g NH4+-N L−1 d−1 were achieved by biofilms enriched with EAB, AOB, and AnAOB, respectively. With influent 444 ± 65 mg NH4+-N d−1, nitrite accumulation between 84 and 105 mg N d−1 was observed independently of the biofilm type. The AnAOB-enriched biofilm released electrons at higher potential energy levels (anode potential of 0.253 V vs. SHE) but had high internal resistance (Rint) of 299 Ω, which limits its power density (0.2 W m−3). For AnAOB enriched biofilm, accumulation of nitrite was a limiting factor for power output by allowing conventional anammox activity without current generation. AOB enriched biofilm had Rint of 18 ± 1 Ω and yielded power density of up to 1.4 W m−3. The activity of the AOB-enriched biofilm was not dependent on the accumulation of dissolved oxygen and achieved 1.5 fold higher coulombic efficiency when sulfate was not available. The EAB-enriched biofilm adapted to oxidize ammonia without organic carbon, with Rint of 19 ± 1 Ω and achieved the highest power density of 11 W m−3. Based on lab-scale experiments (scaling-up factors not considered) energy savings of up to 7 % (AnAOB), 44 % (AOB) and 475 % (EAB) (positive energy balance), compared to conventional nitrification, are projected from the applications of BEAO in wastewater treatment plants.

A strategy for fast anammox biofilm formation under mainstream conditions

One of the bottlenecks to applying anaerobic ammonium oxidation (Anammox) is the long start-up time, especially under mainstream conditions. This study proposed a strategy for fast anammox biofilm formation under mainstream conditions. By first cultivating an aerobic heterotrophic biofilm, and then transferring to anoxic conditions, a pre-cultivated heterotrophic biofilm can be formed in 12 days. The pre-cultivated heterotrophic biofilm then functions as a “glue” to accelerate anammox bacteria adhesion and biofilm formation. Secondary settled effluent with externally added 15–30 mg-N·L−1 ammonium and nitrite was applied as reactor influent. With a single inoculation of suspended growth anammox-laden biomass and no bioaugmentation, an anammox-enriched biofilm formed after 5 months of operation under uncontrolled temperature of 15–20 °C. Both the nitrogen removal rate and specific anammox activity exponentially increased over the course of study, corresponding to an estimated anammox doubling time of 10.8 days. The biofilm thickness on primed carriers was 2–3 times higher than on the non-primed carriers over the first 5 months of operation, and the hszA gene copy number in primed biofilms revealed was consistently 1 to 2 times higher than the non-primed carrier biofilm, indicating that biofil m carrier priming via selection for a pre-cultivated heterotrophic biofilm base can effectively improve the anammox enrichment rate at early stages of reactor operation. Time, rather than the type of biofilm (primed versus non-primed), had a stronger influence on microbial community structure over the full 230 days of reactor operation. Candidatus Brocadia was the only detected anammox bacteria genus. Overall, pre-cultivation of heterotrophs on biofilm carriers provides a simple route to accelerate anammox-enriched biofilm formation under mainstream conditions.

Exploring interactions between quorum sensing communication and microbial development in anammox membrane bioreactor

The long duration required to enrich bacteria that perform anaerobic ammonium oxidation (anammox) limits their wide application in wastewater treatments. Anammox enrichment requires extensive microbial development, but the mechanisms involved are still being studied. Here, a total nitrogen removal rate of 1.22 kg N/m3/day was obtained over 280 days when anammox was enriched in a membrane bioreactor (MBR). Interactions between quorum sensing (QS) and microbial community development were investigated. Acyl-homoserine lactones (AHLs) were detected in the MBR effluent, with C8-HSL having the highest concentration (2.05 ng/L). The specific anammox activity (SAA) of the biomass increased from 335.8 (± 16.4) mg NH4+-N/g VSS/day to 382.9 (± 19.6) mg NH4+-N/g VSS/day with 0.1 μM C8-HSL addition. Anammox bacteria from the genus Candidatus Brocadia were enriched in the MBR. Metagenomic analysis suggested quorum sensing dependent on AHL and c-di-GMP (bis-[3′−5′]-cyclic dimeric guanosine monophosphate) contributed to microbial development. C-di-GMP-dependent quorum sensing may impact the formation of extracellular polymeric substances (EPS) and cell motility of biomass.

Novel synergistic metabolic processes for phenanthrene biodegradation by a nitrate-reducing phenanthrene-degrading culture and an anammox culture

The synergistic metabolism by anammox cultures and nitrate-reducers for anaerobic PAH biodegradation is largely unknown, including whether anammox culture and which kind of anammox bacterium can perform nitrogen metabolism in the anaerobic PAH biodegradation processes, the inhibitory effect of PAH on anammox activity and nitrite on PAH-degrading nitrate-reducers activity, and the synergistic metabolic processes. Herein, an anammox culture that can eliminate nitrite accumulation and decrease inorganic carbon emission during anaerobic phenanthrene (a model of PAH in this study) biodegradation, the synergistic mechanism for phenanthrene biodegradation by a nitrate-reducer and such anammox culture, and the inhibition effect of phenanthrene on such anammox culture and nitrite on a phenanthrene-degrading nitrate-reducer were newly discussed. The results showed that nitrite largely accumulated during anaerobic phenanthrene biodegradation (nitrate accumulation is a common phenomenon for the biodegradation of refractory matter, including PAHs, by nitrate-reducers) by a nitrate-reducer, PheN2, which mineralizes phenanthrene to inorganic carbon, and nitrite was verified as an inhibiting factor for further biodegradation. Anaerobic phenanthrene biodegradation rates and nitrite concentrations (0–7 mM) appeared to have a negative linear correlation. The anammox culture that mainly contained Candidatus Kuenenia was newly found to efficiently reduce nitrite accumulation and inorganic carbon emissions and significantly promote biodegradation efficiency by ∼1.94-fold. Our results showed that phenanthrene absorbed in and on anammox cells had a more direct relationship with the inhibitory effect on anammox activity than phenanthrene in the environment, and 15.2 mg/gVSS phenanthrene absorbed in and on the cells (4 mM concentration in the culture) showed nearly complete inhibition of anammox culture in this study. In addition, few (less than 2% abundance) anammox bacteria were found to be enough for the removal of nitrite produced from anaerobic phenanthrene biodegradation. In an ideal world, co-pollutants of ammonia, nitrate, phenanthrene, and nitrite could be converted to nitrogen gas and biomass by the synergistic metabolism of anammox cultures and nitrate reducers. Our study reveals a new synergistic process that may exist in our environments for PAH elimination by an anammox culture and a nitrate-reducer, which provides a new strategy for the bioremediation of PAH-polluted anoxic zones.

Fast start-up and stable operation of mainstream anammox without inoculation in an A2/O process treating low COD/N real municipal wastewater

It is of great significance to start up the anammox process in the most commonly used anaerobic-anoxic-oxic (A2/O) process in treating mainstream municipal wastewater. Recently, partial-denitrification/anammox (PD/A) has attracted increasing interest as a new avenue in mainstream. This study investigated the in situ start-up of PD/A process in a traditional A2/O process. The PD/A system was rapidly started up within 60 days by adding virgin carriers into the anoxic zone and then run stably for the next 90 days. The in situ anammox activity reached 1.0 ± 0.1 mg NH4+-N/L/h contributing 37.9 ± 6.2% of total nitrogen removal. As a result, the nitrogen removal efficiency of the system increased by 16.9%. The anammox bacteria (AnAOB) on the anoxic biofilms were enriched with a doubling time of 14.53d, and the relative abundance reached 2.49% on day 150. Phylogenetic analysis showed the dominant AnAOB was related to Ca. Brocadia sp. 40, which was the only detected anammox genus in the anoxic biofilm from start-up to stable operation. Batch tests and qPCR results revealed that compared with the floc sludge, the anoxic biofilms exhibited NO2− accumulation driven by PD and performed a better coordination between denitrifiers and AnAOB. Overall, this study provides great confidence for the in situ fast start-up of mainstream anammox using conventional activated sludge.