Abstract ：

In an anammox biofilm reactor, long-term operation inevitably leads to the repeated formation of localized dead zones. Once these dead zones (DZs) occur, the anammox reactor's nitrogen removal efficiency is severely reduced. However, the mechanisms and intrinsic reasons for the transformation of DZs remain unexplored. In this study, the pilot-scale biofilters were classified into biologically active zones (BZs), transition zones (TZs), and DZs. The results indicated that microbial communities undergo accelerated succession from the TZ. Biofilms respond to environmental stress from the DZs by altering the levels of signaling molecules, triggering a series of cascading reactions. These reactions alter the abundance of genes involved in nitrogen removal, promote substance transformation, and speed up the succession of microbial communities. This study demonstrates the objectives and self-healing mechanisms of the anammox biofilm process in the presence of dead zones, which could support the long-term application of anammox technology.

Keyword ：

Biofilm Biofiltration reactor AHLs Quorum sensing Anammox Dead zone

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Abstract ：

Strengthening the activity competitiveness of anaerobic ammonium oxidation (anammox) bacteria (AnAOB) under low nitrogen conditions is indispensable for mainstream anammox application. This study demonstrates that sponge iron addition (42.8 g/L) effectively increased apparent AnAOB activity and extracellular polymeric substance (EPS) production of low load anammox biofilms cultivated under low (influent of 60 mg N/L) and even ultra -low (influent of 10 mg N/L) nitrogen conditions. In -situ batch tests showed that after sponge iron addition the specific AnAOB activity in the low and ultra -low nitrogen systems further increased to 1.18 and 0.47 mmol/g VSS/h, respectively, with an apparent growth rate for AnAOB of 0.011 +/- 0.001 d-1 and 0.004 +/- 0.001 d-1, respectively. The averaged EPS concentration of anammox biofilm in both low (from 35.84 to 71.05 mg/g VSS) and ultra -low (from 44.14 to 57.59 mg/g VSS) nitrogen systems increased significantly, while a higher EPS protein/polysaccharide ratio, which was positively correlated with AnAOB activity, was observed in the low nitrogen system (3.54 +/- 0.34) than that in the ultra -low nitrogen system (1.82 +/- 0.10). In addition, Candidatus Brocadia was detected as dominant AnAOB in the anammox biofilm under the low (12.2 %) and ultra -low (24.7 %) nitrogen condition. Notably, the genus Streptomyces (26.3 %), capable for funge-like codenitrification, increased unexpectedly in the low nitrogen system, but not affecting the nitrogen removal performance. Therefore, using sponge iron to strengthen AnAOB activity under low nitrogen conditions is feasible, providing support for mainstream anammox applications.

Keyword ：

Anammox Biofilm Microbial community Sponge iron Substrate concentrations

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Abstract ：

Combined partial nitrification-denitrification/anammox (PN-PD/A) processes have attracted great attention from researchers in recent years to achieve high nitrogen removal from low carbon /nitrogen (C/N) municipal wastewater. In this context, a step-feed anoxic/oxic (A/O) process was conducted in this study through the combination of the partial nitrification-anammox (PN/A) and partial denitrification-anammox (PD/A) to remove N from municipal wastewater with low C/N. The enhancement of the PN-PD/A process resulted in N removal efficiency of 85.6% at C/N of 2.8. The contributions of the anammox reached 36.4 and 8.8% in the anoxic and oxic chambers, respectively. The biocarriers added to the anoxic and oxic chambers increased the relative abundance of the anammox bacteria in biofilms from 0.61% to 1.51% and 1.02%, respectively. This study demonstrated that employing the step-feed A/O process can create optimal conditions for the anammox bacteria growth, thereby ensuring advanced N removal from low C/N municipal wastewater.

Keyword ：

One-stage anammox process Biocarriers Mainstream Biological nitrogen removal Multistage A/O

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A promising technology for partial nitritation/anammox (PN/A) processes to treat ammonium wastewater is integrated fixed-film activated sludge (IFAS). For practical applications, achieving efficient enrichment of anammox bacteria (AnAOB) remains a challenge. In this study, membranes were temporarily used to separate solid and liquid components to induce changes in the mixed liquor suspended solids of the flocs. With membrane separation, AnAOB proliferated rapidly with a seven-fold increase in the maximum specific growth rate (mu) (from 0.009 to 0.072 d-1) and a three-fold increase in the nitrogen removal rate (from 0.91 to 3.20 kg N/(m3 & sdot;d)). Moreover, microbial community analysis showed significant changes in bacterial species richness and diversity with and without membrane separation. Overall, the regulation of flocs significantly influenced the microbial community structure of both flocs and biofilms leading to improved nitrogen removal efficiency in the IFAS-PN/ A system.

Keyword ：

IFAS Biofilm Autotrophic nitrogen removal Membrane separation Microbial community

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Nitrate or nitrite-dependent anaerobic methane oxidation (n-DAMO) is a microbial process that links carbon and nitrogen cycles as a methane sink in many natural environments. This study demonstrates, for the first time, that the nitrite-dependent anaerobic methane oxidation (nitrite-DAMO) process can be stimulated in sewer systems under continuous nitrate dosing for sulfide control. In a laboratory sewer system, continuous nitrate dosing not only achieved complete sulfide removal, but also significantly decreased dissolved methane concentration by similar to 50 %. Independent batch tests confirmed the coupling of methane oxidation with nitrate and nitrite reduction, revealing similar methane oxidation rates of 3.68 +/- 0.5 mg CH4 L-1 h(-1) (with nitrate as electron acceptor) and 3.57 +/- 0.4 mg CH4 L-1 h(-1) (with nitrite as electron acceptor). Comprehensive microbial analysis unveiled the presence of a subgroup of the NC10 phylum, namely Candidatus Methylomirabilis (n-DAMO bacteria that couples nitrite reduction with methane oxidation), growing in sewer biofilms and surface sediments with relative abundances of 1.9 % and 1.6 %, respectively. In contrast, n-DAMO archaea that couple methane oxidation solely to nitrate reduction were not detected. Together these results indicated the successful enrichment of n-DAMO bacteria in sewerage systems, contributing to approx. 64 % of nitrite reduction and around 50 % of dissolved methane removal through the nitrite-DAMO process, as estimated by mass balance analysis. The occurrence of the nitrite-DAMO process in sewer systems opens a new path to sewer methane emissions.

Keyword ：

Nitrate dosing Nitrite-dependent anaerobic methane oxidation (nitrite-DAMO) Anaerobic methane oxidation Sewer Dissolved methane removal Sulfide control

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Abstract ：

This study constructed an integrated algae/partial nitrification/anammox biofilm system and operated it for 240 days. The total nitrogen removal efficiency exceeded 90 %. The structure, compositions, and function of this symbiotic biofilm, which played a pivotal role in the system, were analyzed in detail. Microscope photos and fluorescence in situ hybridization both showed that bacteria and algae were well integrated. The dissolved oxygen gradient further confirmed that different functional microorganisms grew at varying depths within biofilm. Algae formed an oxygen-producing zone (0-0.48 mm), followed by ammonia oxidizing bacteria (AOB) consuming oxygen to form an oxygen-consuming zone (0.48-0.86 mm), and anaerobic ammonia oxidizing bacteria (AnAOB) removed nitrogen in anaerobic zone (>0.86 mm). Chlorella, Nitrosomonas and Candidatus_Kuenenia were identified as the dominant algae, AOB and AnAOB, with relative abundances of 11.80 %, 19.77 % and 3.07 %, respectively. This layered biofilm benefitted providing a suitable environment for various microorganisms to survive within a complex biofilm.

Keyword ：

Nitrogen Algae Partial nitrification Anammox Biofilm

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Abstract ：

Applying anaerobic ammonium oxidation (anammox) in municipal wastewater treatment plants (MWWTPs) can unlock significant energy and resource savings. However, its practical implementation encounters significant challenges, particularly due to its limited compatibility with carbon and phosphorus removal processes. This study established a pilot-scale plant featuring a modified anaerobic-anoxic-oxic (A(2)O) process and operated continuously for 385 days, treating municipal wastewater of 50 m(3)/d. For the first time, we propose a novel concept of partial denitrifying phosphorus removal coupling with anammox (PDPRA), leveraging denitrifying phosphorus-accumulating organisms (DPAOs) as NO2- suppliers for anammox. N-15 stable isotope tracing revealed that the PDPRA enabled an anammox reaction rate of 6.14 +/- 0.18 mu mol-N/(Lh), contributing 57.4 % to total inorganic nitrogen (TIN) removal. Metagenomic sequencing and 16S rRNA amplicon sequencing unveiled the co-existence and co-prosperity of anammox bacteria and DPAOs, with Candidatus Brocadia being highly enriched in the anoxic biofilms at a relative abundance of 2.46 +/- 0.52 %. Finally, the PDPRA facilitated the synergistic conversion and removal of carbon, nitrogen, and phosphorus nutrients, achieving remarkable removal efficiencies of chemical oxygen demand (COD, 83.5 +/- 5.3 %), NH4+ (99.8 +/- 0.7 %), TIN (77.1 +/- 3.6 %), and PO43- (99.3 +/- 1.6 %), even under challenging operational conditions such as low temperature of 11.7 degrees C. The PDPRA offers a promising solution for reconciling the mainstream anammox and the carbon and phosphorus removal, shedding fresh light on the paradigm shift of MWWTPs in the near future.

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Mainstream anammox Nutrient removal Pilot-scale study Municipal wastewater Denitrifying phosphorus removal

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Abstract ：

Controlling sludge concentration is an effective means to achieve PN. In this article, the reactor used domestic sewage as raw water and promoted the high enrichment of anammox bacteria by controlling the MLVSS of flocs to 1000-1500 mg/L and increasing the concentration of filler sludge. The measures to reduce the concentration of flocculent sludge increased the proliferation rate of the biofilm and provided sufficient substrate for AnAOB. After 102 days of operation, the abundance of Candidatus Brocadia increased from 0.43% during inoculation to 23.56% in phase VI. The ability of the microbial community to utilize energy metabolism and produce ATP was significantly improved, and the appropriate distribution of anammox bacteria and nitrifying, denitrifying bacteria in the ecological niche led to its high enrichment. In summary, this study proposes a strategy to promote the high enrichment of anammox bacteria in mainstream domestic sewage without adding any chemicals.

Keyword ：

Partial nitrification (PN) Anammox bacteria enrichment Control sludge concentration Domestic sewage

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Abstract ：

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

Keyword ：

Multivariable analysis Time -series biofilms Microbial succession Morphological features PN A system

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Abstract ：

Anammox is widely considered as the most cost-effective and sustainable process for nitrogen removal. However, how to achieve the enrichment of anammox biomass remains a challenge for its large-scale application, especially in mainstream wastewater treatment. In this study, the feasibility of enrichment of anammox biomass was explored through the realization of partial denitrification and the addition of bio-carriers. By using ordinary activated sludge, a sequencing batch reactor (SBR) followed by an up-flow anaerobic sludge bed (UASB) was operated at 25 +/- 2 degrees C for 214 days. The long-term operation was divided into five phases, in which SBR and UASB were started-up in Phases I and II, respectively. By eliminating oxygen and adjusting the inflow ratios in Phases III-V, advanced nitrogen removal was achieved with the effluent total nitrogen being 4.7 mg/L and the nitrogen removal efficiency being 90.5% in Phase V. Both in-situ and ex-situ activity tests demonstrated the occurrence of partial denitrification and anammox. Moreover, 16S rRNA high-throughput sequencing analysis revealed that Candidatus Brocadia was enriched from below the detection limit to in biofilms (0.4% in SBR, 2.2% in UASB) and the floc sludge (0.2% in SBR, 1.3% in UASB), while Thauera was mainly detected in the floc sludge (8.1% in SBR, 8.8% in UASB), which might play a key role in partial denitrification. Overall, this study provides a novel strategy to enrich anammox biomass driven by rapid achievement of partial denitrification through the addition of bio-carriers, which will improve large-scale application of anammox processes in mainstream wastewater treatment. (c) 2023 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.

Keyword ：

Partial denitrification Enrichment Bio-carriers Anammox biomass Mainstream wastewater

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