Biological Nitrogen Removal Research Articles

Genome-resolved metagenomics reveals the nitrifiers enrichment and species succession in activated sludge under extremely low dissolved oxygen

Nitrification, a process carried out by aerobic microorganisms that oxidizes ammonia to nitrate via nitrite, is an indispensable step in wastewater nitrogen removal. To facilitate energy and carbon savings, applying low dissolved oxygen (DO) is suggested to shortcut the conventional biological nitrogen removal pathway, however, the impact of low DO on nitrifying communities within activated sludge is not fully understood. This study used genome-resolved metagenomics to compare nitrifying communities under extremely low- and high-DO. Two bioreactors were parallelly operated to perform nitrification and DO was respectively provided by limited gas-liquid mass transfer from the atmosphere (AN reactor, DO < 0.1 mg/L) and by sufficient aeration (AE reactor, DO > 5.0 mg/L). Low DO was thought to limit nitrifiers growth; however, we demonstrated that complete nitrification could still be achieved under the extremely low-DO conditions, but with no nitrite accumulation observed. Kinetic analysis showed that after long-term exposure to low DO, nitrifiers had a higher oxygen affinity constant and could maintain a relatively high nitrification rate, particularly at low levels of DO (<0.2 mg/L). Community-level gene analysis indicated that low DO promoted enrichment of nitrifiers (the genera Nitrosomonas and Nitrospira, increased by 2.3- to 4.3-fold), and also harbored with 2.3 to 5.3 times higher of nitrification functional genes. Moreover, 46 high-quality (>90 % completeness and <5 % contamination) with 3 most abundant medium-quality metagenome-assembled genomes (MAGs) were retrieved using binning methods. Genome-level phylogenetic analysis revealed the species succession within nitrifying populations. Surprisingly, compared to DO-rich conditions, low-DO conditions were found to efficiently suppressed the ordinary heterotrophic microorganisms (e.g., the families Anaerolineales, Phycisphaerales, and Chitinophagales), but selected for the specific candidate denitrifiers (within phylum Bacteroidota). This study provides new microbial insights to demonstrate that low-DO favors the enrichment of autotrophic nitrifiers over heterotrophs with species-level successions, which would facilitate the optimization of energy and carbon management in wastewater treatment.

Ammonia oxidizing bacteria (AOB) denitrification and bacterial denitrification as the main culprit of high N2O emission in SBR with low C/N ratio wastewater

A large amount of greenhouse gas nitrous oxide (N2O) will be produced during the biological nitrogen removal process for organic wastewater of low C/N ratio. One of the effective methods to solve this problem is to incorporate inexpensive carbon source. In this study, raw wastewater (RW) from pig farm, that was not anaerobically digested, was utilized as exogenous carbon in both A/O and SBR aerobic reactor to treat liquid digestate with high ammonia nitrogen and low C/N ratio. The results showed that N2O emission in SBR was higher than that of A/O process under the same nitrogen load. The N2O conversion in the biological nitrogen removal process was investigated by the strategy of integrating stable isotope method and metagenomics. The δO18-N2O, δN15-N2O, and SP values of the SBR were closer to the denitrification values of Ammonia-Oxidizing Bacteria (AOB) than those of A/O. The abundance of AOB in the SBR reactor was higher than that in the A/O reactor, while the abundance of denitrifying bacteria was lower. The amoA/B/C gene abundance in the SBR was greater than that in the A/O, and the NOS gene abundance was the opposite. The results indicated that both AOB denitrification and bacterial denitrification led to the increase of N2O emissions of the SBR.

Thauera enriched granular activated carbon-assisted aerobic granular sludge performs enhanced biological nitrogen and phosphate removal via propionate utilization

Propionate constituting up to 25 % of volatile fatty acids in municipal wastewater, presents metabolic challenges for microorganisms. This study investigated the combined use of propionate along with granular activated carbon (GAC) for aerobic granular sludge (AGS) development and biological nutrient removal (BNR), particularly bio-P removal in granular reactors. GAC addition decreased granulation time and facilitated stable BNR pathways with propionate. AGS demonstrated robust settling properties (≥1 mm, 6 g/L MLSS, 40 mL/g SVI) with increased alginate-like exopolysaccharide (ALE) content up to 400 mgALE/gSS, indicating stable granule formation. Effective ammonium removal via simultaneous nitrification and denitrification was achieved. By day 90, phosphate removals reached 89 %, due to enrichment of polyphosphate accumulating organisms (PAOs) in GAC-containing AGS. The sludge fraction (>0.5 mm) comprised of granules and GAC-biofilms showed efficient phosphate removal through enhanced biological phosphate removal (EBPR). Enrichment of denitrifying PAO, Thauera sp., contributed to enhanced granulation, granular stability and nutrient removals.

Enhanced nitrogen removal from low C/N municipal wastewater through nitrification, partial denitrification and anammox via a step-feed strategy: Synergistic contribution by bacteria in biofilm and floc sludge

Energy-efficient nitrogen removal from carbon-limited municipal wastewater poses challenges. This study developed a novel partial denitrification and anammox (PD/A) process using a step-feed strategy in a single-stage integrated fixed-biofilm activated sludge (IFAS) system operated under sequencing batch mode. Despite relatively low temperatures (15.9–21.6 °C), biological nitrogen removal efficiency reached 80.5 % without the addition of external carbon sources. With the total inorganic nitrogen (TIN) of 48.9 ± 1.2 mg N/L, and C/N of 4.0 in influent, the TIN in effluent achieved 9.6 ± 1.5 mg N/L. Anammox bacteria (AnAOB) activity in the biofilm was 27.96 mg N/(g VSS·d), which was threefold greater than in floc sludge. Candidatus Brocadia (AnAOB) presented a high relative abundance in biofilm (0.53 %), whereas denitrifying bacteria including Thauera and Candidatus Competibacter were mainly retained in floc sludge. Overall, the coupling of step-feed strategy and IFAS offers novel insights into the efficient nitrogen removal from low C/N domestic sewage for retrofitting of municipal wastewater treatment plants, not only saving the energy consumption for aeration, but also fully utilizing carbon sources and ammonium in the influent for the PD/A process.

Metabolic evolution and bottleneck insights into simultaneous autotroph-heterotroph anammox system for real municipal wastewater nitrogen removal

When biological nitrogen removal (BNR) systems shifted from treating simulated wastewater to real wastewater, a microbial succession occurred, often resulting in a decline in efficacy. Notably, despite their high nitrogen removal efficiency for real wastewater, anammox coupled systems operating without or with minimal carbon sources also exhibited a certain degree of performance reduction. The underlying reasons and metabolic shifts within these systems remained elusive. In this study, the simultaneous autotrophic/heterotrophic anammox system demonstrated remarkable metabolic resilience upon exposure to real municipal wastewater, achieving a nitrogen removal efficiency (NRE) of 82.83 ± 2.29 %. This resilience was attributed to the successful microbial succession and the complementary metabolic functions of heterotrophic microorganisms, which fostered a resilient microbial community. The system's ability to harness multiple electron sources, including NADH oxidation, the TCA cycle, and organics metabolism, allowed it to establish a stable and efficient electron transfer chain, ensuring effective nitrogen removal. Despite the denitrification channel's nitrite supply capability, the analysis of the interspecies correlation network revealed that the synergistic metabolism between AOB and AnAOB was not fully restored, resulting in selective functional bacterial and genetic interactions and the system's PN/A performance declined. Additionally, the enhanced electron affinity of PD increased interconversion of NO3−-N and NO2−-N, limiting the efficient utilization of electrons and thereby constraining nitrogen removal performance. This study elucidated the metabolic mechanism of nitrogen removal limitations in anammox-based systems treating real municipal wastewater, enhancing our understanding of the metabolic functions and electron transfer within the symbiotic bacterial community.

Effects of gradual increase of ciprofloxacin and cefotaxime on nitrogen and phosphorus removal and microbial community in moving bed biofilm reactor

To clarify the effects of trace amount of antibiotics in wastewater on biofilm reactor, this study explored the effects of gradual increase of ciprofloxacin (CIP) and cefotaxime (CTX) ranging from 100 to 1000 ng·L−1 on the nitrogen and phosphorus removal and microbial community in a moving bed biofilm reactor (MBBR). Under different phases of CIP and CTX gradual increase, the resilience of culturable functional bacteria including ammonifying bacteria, nitrite oxidizing bacteria, aerobic denitrifying bacteria and phosphorus accumulating organisms in the biofilm was analyzed. High-throughput sequencing analysis was used to reveal the microbial community structure and species composition of functional bacteria. The results indicated that the treatment performance of MBBR was temporarily affected by trace CIP and CTX and returned to its original level within 1–2 days. Ammonifying bacteria exhibited strong resilience in trace CIP and CTX stress environments. Trace CIP and CTX altered the microbial community structure in biofilm, but the performance of nitrogen and phosphorus removal in MBBR was slightly affected. This might attribute to the rapid proliferation of Thauera, Dechloromonas and Candidatus_Accumulibacter on the carrier biofilm. The research results help to deepen the understanding of the impact of trace antibiotics on the MBBR and to provide a reference for the development of microbial germplasm resources for biological nitrogen and phosphorus removal from wastewater under antibiotic stress conditions.

Starvation resilience in anammox-based bioreactors: A stable nitrogen removal route on partial denitrification/anammox (PD/A)

This study investigates the performance, resilience and microbial community dynamics of two anaerobic processes, i.e. pure anammox (R1) and partial denitrification/anammox (PD/A) (R2), following a 30-day starvation period. The tolerance to starvation was assessed by comparing nitrogen removal efficiency and microbial activity across both reactors. Results show that the PD/A process recovery to pre-starvation performance levels within just one day, as compared to the pure anammox process. Notably, although the activity of anammox bacteria decreased in both processes during starvation, the decay rate in R1 was 69.59 % higher than in R2, potentially explaining the quicker recovery of R2. Furthermore, enhanced secretion of extracellular polymeric substance (EPS) during starvation served as a protective mechanism. The potential functions and genes in microorganisms, as well as the pathway of nitrogen cycling, were demonstrated through analyses using the KEGG database. This research reveals essential mechanistic insights and strategic guidance for the effective implementation of anammox-based biological nitrogen removal processes.

Effect of elemental sulfur on anaerobic ammonia oxidation: Performance and mechanism

Biological nitrogen removal processes provide effective means to mitigate nitrogen-related issues in wastewater treatment. Previous studies have highlighted the collaborative efficiency between sulfur autotrophic denitrification and Anammox processes. However, the trigger point induced the combination of nitrogen and sulfur metabolism is unclear. In this study, elemental sulfur (S0) was introduced to Anammox system to figure out the performance and mechanism of S0-mediated autotrophic denitrification and Anammox (S0SAD-A) systems. The results showed that the nitrogen removal performance of the Anammox reactor decreased with the increasing concentrations of NH4+-N and NO2−-N in influent, denitrification occurred when NH4+-N concentration reached 100 mg/L. At stage ⅳ (150 mg/L NH4+-N), the total nitrogen removal efficiency in S0SAD-A system (95.99%) was significantly higher than that in the Anammox system (77.22%). Throughout a hydraulic retention time, the consumption rate of NH4+-N in S0SAD-A was faster than that in Anammox reactor. And there existed a nitrate-concentration peak in S0SAD-A system. Metagenomic sequencing was performed to reveal functional microbes as well as key genes involved in sulfur and nitrogen metabolism. The results showed that the introduction of S0 elevated the abundance of Ca. Brocadia. Moreover, the relative abundance of Anammox genes, such as hao, hzsA and hzsC were also stimulated by sulfur. Notably, unclassified members in Rhodocyclaceae acted as the primary contributor to key genes involved in the sulfur metabolism. Overall, the interactions between Anammox and denitrification were stimulated by sulfur metabolism. Our study shed light on the potential significance of Rhodocyclaceae members in the S0SAD-A process and disclosed the relationship between anammox and denitrification.

Enhanced-nitrogen removal through Fe(III)-triggered partial dissimilatory nitrate reduction to ammonium coupling with anammox in anammox bioreactor

Anammox is recognized as a prospective alternative for future biological nitrogen removal technologies. However, the nitrate by-products produced by anammox bacteria limit its overall nitrogen removal efficiency below 88 %. This study introduced Fe(III) into the anammox bioreactor to enhance the nitrogen removal efficiency to approximately 95 %, surpassing the biochemical limit of 88 % imposed by anammox stoichiometry. Anammox sludge was demonstrated to utilize extracellular polymeric substances to reduce Fe(III) into Fe(II), and this process promoted the dominance of Ca. Brocadia. The iron addition improved the abundance of narGHI genes and facilitated the partial dissimilatory nitrate reduction to ammonium, with nitrite as the end product. The accumulated nitrite was then eliminated through the anammox pathway, along with the excess ammonium (30 mg/L) in the influent. Overall, this study deepens our understanding of the enhanced nitrogen removal triggered by Fe(III) in anammox sludge and offers an effective approach to boost anammox process.

Impacts of cefalexin on nitrite accumulation, antibiotic degradation, and microbial community structure in nitrification systems

The intensive use of various antibiotics for clinical and agricultural purposes has resulted in their widespread use in wastewater treatment plants. However, little research has been conducted on the effects of antibiotics on nitrite accumulation, antibiotic degradation pathways, or the microbial community structure in nitrification systems. In this study, a laboratory-scale sequencing batch reactor was used to treat wastewater containing cefalexin (CFX) at different doses (5, 10, 15, and 20 mg/L). The results showed that the nitrification performance was gradually inhibited with increasing CFX concentration. Ammonia-oxidizing bacteria (AOB) are more tolerant to CFX than nitrite-oxidizing bacteria (NOB). Under 15 mg/L of CFX, NOB were completely suppressed, whereas AOB were partially inhibited, as evidenced by an ammonium removal efficiency of 60 % and a 90 % of nitrite accumulation ratio. The partial nitritation was achieved. CFX can be degraded into 2-hydroxy-3phenylpyrazine and cyclohexane through bacterial co-metabolism, and CFX degradation gradually diminishes with decreasing nitrification performance. The abundance of Nitrospira gradually decreased with increasing CFX concentration. Ferruginibacter, Hydrogenophaga, Thauera, and Pseudoxanthomonas were detected at relative abundances of 13.2 %, 0.4 %, 0.9 %, and 1.3 %, respectively, indicating their potential roles in antibiotic degradation. These findings provide insight into the interactions between antibiotics and microbial communities, which are beneficial for a better understanding of antibiotic degradation in nitrification systems.

Firmicutes and Bacteroidetes as the dominant microorganisms for ammonium nitrogen wastewater treatment with a low C/N ratio in BCOR

Improving nitrogen removal is a significant concern in the biological treatment of ammonium nitrogen (NH4+-N) wastewater with low chemical oxygen demand (COD)/NH4+-N (C/N) ratio, and microorganisms are the critical factor affecting nitrogen removal. In this study, a laboratory-scale biological contact oxidation reactor (BCOR) system was successfully started up (day 1 to day 47) and stabilized (day 48 to day 320). A 16S rRNA gene sequencing analysis showed that the phyla Firmicutes and Bacteroidetes were first observed as the dominant microorganisms in the BCOR for the ammonium nitrogen wastewater treatment with a low C/N ratio. The genera Bacteroides (phylum Bacteroidetes), Porphyromonas (phylum Bacteroidetes), Dialister (phylum Firmicutes), and Anaerococcus (phylum Firmicutes) also became the main microorganisms in this system for the first time. These microbial communities enabled the system to achieve an average ammonium nitrogen removal efficiency and total nitrogen removal efficiency of 95.8 % and 88.1 %, respectively, during the operational period. It indicated that these dominant microorganisms in the BCOR system played an essential role in improving the performance of biological nitrogen removal. This conclusion will play a critical role in promoting the microbial mechanism research and technology engineering application of the BCOR system for treating ammonium nitrogen wastewater with a low C/N ratio.

Microbial response to the chronic toxicity effect of graphene and graphene oxide nanomaterials within aerobic granular sludge systems

Nanomaterials present in wastewater can pose a significant threat to aerobic granular sludge (AGS) systems. Herein, we found that compared to graphene nanomaterials (G-NMs), the long-term presence (95 days) of graphene oxide nanomaterials (GO-NMs) resulted in an increased proliferation of filamentous bacteria, poorer sedimentation performance (SVI30 of 74.1 mL/g) and smaller average particle size (1224.4 µm) of the AGS. In particular, the GO-NMs posed a more significant inhibitory effect to the total nitrogen removal efficiency of AGS (decreased by 14.3 %), especially for the denitrification process. The substantial accumulation of GO-NMs within the sludge matrix resulted in a higher level of reactive oxygen species in AGS compared to G-NMs, thereby inducing lactate dehydrogenase release, and enhancing superoxide oxidase and catalase activities. Such excessive oxidative stress could potentially result in a significant reduction in the activity of nitrogen metabolism enzymes (e.g., nitrate reductase and nitrite reductase) and the expression of key functional genes (e.g., nirS and nirK). Altogether, compared to G-NMs, prolonged exposure to GO-NMs had a more significant chronic toxicity effect on AGS systems. These findings implied that the presence of G-NMs and GO-NMs is a hidden danger to biological nitrogen removal and should receive more attention.

Beyond denitrification: Alginate production by mucoid Pseudomonas aeruginosa using wastewater nitrate as electron acceptor

Microbial synthesis is an effective method for carbon and nitrogen recovery in the biological nitrogen removal process. The recycling of alginate-like exopolymers has attracted the attention of many researchers; however, the composition of the polymers and alginate differs. In this study, the capacity and mechanism of a mucoid Pseudomonas aeruginosa to remove nitrate and synthesize alginate were investigated. The results showed that the P. aeruginosa achieved higher nitrate removal efficiency (95.3 %) and alginate yield (298 mg/L) after 36 hours of microaerobic cultivation compare to aerobic condition. The optimal conditions for nitrogen removal and alginate recovery are 35 °C, pH 7 and C/N 15. The transcriptional activities of denitrifying genes in P. aeruginosa were upregulated at the low dissolved oxygen level. Although O2 may upregulate the expression of alginate synthesis key genes (algD, alg8 and alg44) by activating AlgZ-AlgR two-component signal transduction system, microaerobic condition facilitates P. aeruginosa proliferation and results in a higher alginate yield. Moreover, the P. aeruginosa strain was further coupled with an anoxic/aerobic reactor for deeper nitrogen removal, and the nitrate removal efficiency and alginate yield were maintained above 91 % and 100 mg/L, respectively. The conversion efficiency of sodium acetate to alginate reached 15 %. This work provides an economically and environmentally sustainable strategy for wastewater treatment and the recovery of high-value bioresource.

Optimization of biological nitrogen removal in full-scale municipal WWTPs using activated sludge model simulation

The study evaluated the most efficient biological nitrogen removal (BNR) process in four full-scale municipal wastewater treatment plants (WWTPs) by using BioWin, a simulation software based on the activated sludge model (ASM). A series of experiments were conducted to determine the kinetic and stoichiometric parameters for the ASM. Results indicated that autotrophic maximum specific growth rates in the studied WWTPs were generally higher compared to previous findings, likely due to their low COD/N ratios, emphasizing the importance of local parameterization. Continuous water quality monitoring in each plant was employed to validate the model. Dynamic simulation results indicated that the error remained within an acceptable range, with a mean relative error of less than 20%, confirming the reliability of ASM parameters derived from batch experiments. Subsequently, various operational scenarios were analyzed to determine the optimal BNR process for each plant, considering influent flow rate, internal recycling, and methanol addition. Simulation outcomes suggested that O/A (oxic/anoxic) operation is preferable for plants with low organic content (COD = 60–110 mg/L), considering both operational costs and total nitrogen removal rate. Conversely, A/O (anoxic/oxic) operation might be advantageous for plants with higher influent organic matter (COD = 200 mg/L).

Enhancement of Biological Nitrogen Removal System Resilience to Chronic Exposure of Zinc Oxide Nanoparticles by Quorum Sensing Modulation: Physiochemical, Microbial, and Metabolic Insights

The effects of three typical N-acyl-homoserine lactones (AHLs) on the tolerance of biological nitrogen removal (BNR) system to chronic exposure of zinc oxide nanoparticles (NPs) were investigated. C4-HSL successfully delayed the crash time of nitrogen removal performances in the NP-stressed system, while C6-HSL and C10-HSL maintained total nitrogen removal efficiencies throughout the 90-day NP exposure. All three AHLs increased NPs’ contents captured in extracellular polymeric substances, alleviating membrane damage and preserving floc structure. The activities of tricarboxylic acid cycle-related enzymes and the relative abundances of BNR-related functional genes and genera were significantly enhanced. Besides, C6-HSL and C10-HSL augmented antioxidant enzyme activities and the abundances of functional genes and metabolites related to antioxidation, flagellar assembly, and chemotaxis, which synergistically reduced the reactive oxygen species’ excessive accumulation. The tested AHLs effectively enhanced BNR systems’ tolerance to chronic NP exposure, providing inspiration for quorum sensing applications in emerging contaminant removal.

Ultrasound-induced structural changes in partial nitrification sludge: Unravelling the mechanism for improved nitrogen removal

Low-intensity ultrasound, as a form of biological enhancement technology, holds significant importance in the field of biological nitrogen removal. This study utilized low-intensity ultrasound (200 W, 6 min) to enhance partial nitrification and investigated its impact on sludge structure, as well as the internal relationship between structure and properties. The results demonstrated that ultrasound induced a higher concentration of nitrite in the effluent (40.16 > 24.48 mg/L), accompanied by a 67.76% increase in the activity of ammonia monooxygenase (AMO) and a 41.12% increase in the activity of hydroxylamine oxidoreductase (HAO), benefiting the partial nitrification. Based on the extended Derjaguin–Landau–Verwey–Overbeek (XDLVO) theoretical analysis, ultrasonic treatment enhanced the electrostatic interaction energy (WR) between sludge flocs, raising the total interaction energy from 46.26 kT to 185.54 kT, thereby causing sludge dispersion. This structural alteration was primarily attributed to the fact that the tightly bonded extracellular polymer (TB-EPS) after ultrasound was found to increase hydrophilicity and negative charge, weakening the adsorption between sludge cells. In summary, this study elucidated that the change in sludge structure caused by ultrasonic treatment has the potential to enhance the nitrogen removal performance by partial nitrification.

Ammonia monooxygenase-mediated cometabolic biotransformation of volatile 4-chlorophenol in nitrifying counter-diffused biofilms: A combined molecular dynamics simulation, DFT calculation and experimental study

Ammonia monooxygenase (AMO)-mediated cometabolism of organic pollutants has been widely observed in biological nitrogen removal process. However, its molecular mechanism remains unclear, hindering its practical application. Furthermore, conventional nitrification systems encounter significant challenges such as air pollution and the loss of ammonia-oxidizing bacteria, when dealing with wastewater containing volatile organic pollutants. This study developed a nitrifying membrane-aerated biofilm reactor (MABR) to enhance the biodegradation of volatile 4-chlorophenol (4-CP). Results showed that 4-CP was primarily removed via Nitrosomonas nitrosa-mediated cometabolism in the presence of NH4+-N, supported by the increased nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP) content, AMO activity and the related genes abundance. Hydroquinone, detected for the first time and produced via oxidative dechlorination, as well as 4-chlorocatechol was primary transformation products of 4-CP. Nitrosomonas nitrosa AMO structural model was constructed for the first time using homology modeling. Molecular dynamics simulation suggested that the ortho-carbon in the benzene ring of 4-CP was more prone to metabolismcompared to the ipso-carbon. Density functional theory calculation revealed that 4-CP was metabolized by AMO via H-abstraction-OH-rebound reaction, with a significantly higher rebound barrier at the ipso-carbon (16.37 kcal·mol-1) as compared to the ortho-carbon (6.7 kcal·mol-1). This study fills the knowledge gap on the molecular mechanism of AMO-mediated cometabolism of organic pollutants, providing practical and theoretical foundations for improving volatile organic pollutants removal through nitrifying MABR.

Rapid Start-up of Continuous Autotrophic Nitrogen Removal Reactor by Hybrid-inoculating PN and PN/A Granular Sludges

The rapid cultivation of partial nitritation/ANAMMOX （PN/A） granular sludge in a continuous-flow mode is one of the key technologies for efficient biological nitrogen removal in domestic wastewater treatment. Compared with that in PN/A granular sludge, PN granular sludge demonstrates a shorter incubation period and suitability for batch culture. It is also a good carrier for enriching ANAMMOX （AMX） bacteria. In this study, we established a continuous-flow autotrophic nitrogen removal process in three continuously stirred tank reactors （CSTR） （R1-R3） by hybrid-inoculating PN/A and PN granular sludge at the mass ratios of 3∶1, 1∶1, and 1∶3, respectively. By implementing high ammonium nitrogen loading and short hydraulic retention time, continuous autotrophic nitrogen removal processes were successfully started up in the three CSTRs. The results showed that compared with that of R1 and R2, R3 had a longer start-up time but a similar steady-state nitrogen removal performance. The total nitrogen removal load of R3 could be more than 2.6 kg·（m3·d）-1. Intriguingly, the inoculated PN granular sludge served as a precursor for PN/A granular sludge cultivation. This approach facilitated the enrichment of anaerobic ammonia-oxidizing bacteria （AMX） by introducing abundant ammonium-oxidizing bacteria （AOB） and nitrite nitrogen substrates into the CSTR. According to the results of high-throughput sequencing, the microbial abundance and diversity of the mature granules in R1-R3 were significantly higher than those of the inoculation sludge. AOB （genus Nitrosomonas）, AMX （genera Candidatus Kuenenia and Candidatus Brocadia）, and symbiotic heterotrophs, such as Chloroflexi, Bacteroidetes, and Chlorobi, drove the autotrophic nitrogen removal process and maintained the stability of the granular structure. In summary, a novel start-up strategy of hybrid-inoculating granular sludge was provided for a continuous-flow autotrophic nitrogen removal in engineering application.

Development of an integrated fixed-film activated sludge (IFAS) reactor treating landfill leachate for the biological nitrogen removal through nitritation-denitritation

The current work investigated the performance of an Integrated Fixed-Film Activated Sludge Sequencing Batch Reactor (IFAS-SBR) for Biological Nitrogen Removal (BNR) from mature landfill leachate through the nitritation-denitritation process. During the experimental period two IFAS-SBR configurations were examined using two different biocarrier types with the same filling ratio (50%). The dissolved oxygen (DO) concentration ranged between 2 and 3 mg/L and 4–6 mg/L in the first (baseline-IFAS) and the second (S8-IFAS) setup, respectively. Baseline-IFAS operated for 542 days and demonstrated a high and stable BNR performance maintaining a removal efficiency above 90% under a Nitrogen Loading Rate (NLR) up to 0.45 kg N/m3-d, while S8-IFAS, which operated for 230 days, was characterized by a limited and unstable BNR performance being unable to operate sufficiently under an NLR higher than 0.20 kg N/m3-d. It also experienced a severe inhibition period, when the BNR process was fully deteriorated. Moreover, S8-IFAS suffered from extensive biocarrier stagnant zones and a particularly poor sludge settleability. The attached biomass cultivated in both IFAS configurations had a negligible content of nitrifying bacteria, probably attributed to the insufficient DO diffusion through the biofilm, caused by the low DO concentration in the liquid in the baseline case and the extensive stagnant zones in the S8-IFAS case. As a result of the high biocarrier filling ratio, the S8-IFAS was unstable and low. This was probably attributed to the mass transfer limitations caused by the biocarrier stagnant zones, which hinder substrate and oxygen diffusion, thus reducing the biomass activity and increasing its vulnerability to inhibitory and toxic factors. Hence, the biocarrier filling fraction is a crucial parameter for the efficient operation of the IFAS-SBR and should be carefully selected taking into consideration both the media type and the overall reactor configuration.

An aeration requirements calculating method based on BOD5 soft measurement model using deep learning and improved coati optimization algorithm

Conventionally, the supply-side is over-aerated to meet the oxygen demand of biological wastewater treatment in wastewater treatment plants (WWTPs). Such systems do not consider the various influent loadings and actual oxygen consumption. It cannot respond effectively to fluctuating influent, resulting in unnecessary aeration and energy wastage. In this study, we developed a novel approach to calculate aeration requirements in real time. Firstly, the oxygen demand formula for biological nitrogen and phosphorus removal is modified by considering realistic factors (temperature, oxygen partial pressure and so on) to construct the aeration demand formula. Then, to address the problem that BOD5 in aeration formula is difficult to measure in real time, a novel hybrid model integrating Fuch mapping, reverse learning strategy, Coati Optimization Algorithm (COA), and Backpropagation Artificial Neural Network (BP-ANN) is proposed in this study. Finally, aeration requirements are calculated based on real-time water quality data. Experimental results show that the ICOA-BP model with 9 neurons in hidden layer predicted the optimum BOD5 concentration by achieving a 25.146 mean absolute error and a 0.735 coefficient of determination (all-R2) in all datasets. The optimization led to a 15.164 % aeration savings and 38.942 % energy savings. The proposed method of aeration calculation is automated and intelligent, responding to fluctuating changes in influent quality and reducing unnecessary aeration and energy consumption while meeting the requirements of effluent quality.