Nitrogen Removal Research Articles

Enhancing biological nitrogen removal of slaughterhouse and meat processing wastewater in three-stage AO process by influent allocation: From lab-scale to full-scale investigation

Three-stage anoxic/aerobic (AO) process is promising for efficient nitrogen removal from high-strength wastewater. Herein, the effects of influent allocation modes (50/40/10 vs. 50/25/25) on nitrogen removal performance for treating slaughterhouse and meat processing wastewater (SMPW) were investigated from lab-scale to full-scale three-stage AO processes. Compared to 50/25/25 (94.1 %), the allocation mode of 50/40/10 achieved a higher nitrogen removal of 97.7 % and better adaptability to ammonia load. The second anoxic tank contributed the highest total nitrogen (TN) removal in the system with allocation mode of 50/40/10, while simultaneous nitrification–denitrification and aerobic denitrification in the thrid oxic tank effectively enhanced the TN removal efficiency. Microbial community analysis revealed that SMPW treatment systems tended to enrich lipolytic bacteria (Pseudomonas and Bacillus), cellulolytic bacteria (Christensenellaceae_R-7_group), proteolytic bacteria (Proteiniclasticum, Planococcus, and OLB8), and denitrifying glycogen accumulating bacteria (Candidatus_Competibacter). Meanwhile, the inherent nature of SMPW promoted an increase in the dissimilatory nitrate reduction to ammonium pathway, while the abundant and complex organic substances environment ensured efficient nitrogen removal. The influent allocation mode had no significant effect on nitrification process, but the 50/40/10 allocation mode obviously enriched the nitrous oxide reductase encoded by gene nosZ, which reduced carbon emission. The reliability of 50/40/10 was verified in a 35,000 m3/d full-scale SMPW treatment plant, with the effluent COD, TN, and ammonia nitrogen stabilized at 18 ± 5, 4.7 ± 1.8, and 0.9 ± 0.2 mg/L, respectively, after 48 days. This study provides a valuable reference for effective regulation of three-stage AO process in treating SMPW.

Activated carbon and anthraquinone-2,6-disulfonate as electron shuttles for enhancing carbon and nitrogen removal from simultaneous methanogenesis, Feammox and denitrification system.

Anthraquinone-2,6-disulfonate (AQDS) and activated carbon (AC) were employed as exogenous electron shuttles (ESs) for enhancing the performance of an integrated simultaneous methanogenesis, Feammox, and denitrification (SMFD) system treating fish sludge. The addition of AQDS and AC led to an increased total nitrogen removal efficiency by 30.2% and 66.5%, an increased total chemical oxygen demand removal efficiency by 9.5% and 24.5%, and an improved methane yield by 5.2% and 12.6%, respectively. Regarding nitrogen removal, AQDS mainly facilitated NH4+-N oxidation into NO3--N via Feammox, while AC facilitated both Feammox and denitrification. Regarding carbon removal, both ESs promoted the hydrolysis-acidification process via stimulating dissimilatory iron reduction and established direct interspecies electron transfer (DIET) between methanogens and syntrophic bacteria. Microbial analysis confirmed the enrichment of iron-reducing bacteria, denitrifiers, DIET-related methanogens and syntrophic partners in the presence of ESs. The study provides an ESs-assisted strategy for enhancing simultaneous nitrogen and carbon removal from high-strength wastewater.

Simultaneous removal of organic matter and inorganic nitrogen in Baijiu wastewater by methanotrophic denitrification.

Methanotroph could facilitate nitrogen removal during methane oxidation, and promote conversion of organic compounds by producing methane monooxygenase. Co-metabolic effect and mechanism of aerobic methane oxidation on the removal of nitrogen and organic matter from Baijiu wastewater were investigated using an improved denitrifying biological filter. It was found that the average removal efficiency of chemical oxygen demand (COD), total nitrogen (TN) and chroma increased by 17%, 22% and 10% in reactor B with methane compared to reactor A with air only. Three-dimensional fluorescence spectroscopy and Fourier transform infrared spectroscopy analysis revealed that methanotroph co-metabolism was accompanied by eliminating nitrogen and organic matter as well as forming alcohol compounds. Metagenomic analyses revealed that Methylocaldum, the dominant genera in Reactor B, exerted a pivotal role in removing nitrogen and organic matter removal by supplying energy and catalysis. Functional genes pmoABC-amoABC could facilitate nitrogen and organic matter removal.

Assessment of reactor configurations and key factors for enhanced anammox-based nitrogen removal.

Wastewater treatment processes are continually evolving to meet stringent environmental standards while optimizing energy consumption and operational costs. With significant advantages over more traditional approaches, the anammox process has become a hopeful substitute for nitrogen removal. The objective of this work was to evaluate upflow anaerobic sludge blanket reactor (UASBR), moving bed biofilm reactor (MBBR), and sequential batch reactor (SBR) among diverse reactor configurations, in culturing anammox bacteria and achieving nitrogen removal efficiencies. Synthetic wastewater containing NH4+-N concentration and NO2--N concentration of 80±5mg/L was introduced to the reactors, and observations were made for up to 150 days. This study found that the MBBR demonstrated superior anammox activity, achieving a total nitrogen removal efficiency (TNRE) of 94±3%, SBR exhibited a TNRE of approximately 85±3%, while UASB displayed TNRE of 73±3%. The effect of varying carbon-to-nitrogen (C/N) ratios on nitrogen removal efficiencies was investigated, revealing a decrease in TNRE as the C/N ratio increased from 3 to 8. This study demonstrated the enhancing and inhibitory effects of C/N ratio, NO₂--N, and Fe concentrations. It revealed that Fe concentrations between 1 and 5mg/L increase specific anammox activity (SAA), while concentrations between 5 and 10mg/L negatively impact it. Additionally, NO₂--N concentrations above 150mg/L significantly reduce SAA. Furthermore, a 16S rRNA metagenomic analysis of MBBR sludge samples revealed the significant presence of Candidatus Brocadia bacteria, constituting 20.4% of the microbial community. This research highlights the potential of MBBR in fostering anammox reactions and achieving efficient nitrogen removal in wastewater treatment applications.

Rethinking the effect of salinity on nitrogen removal capacity of aerobic granular sludge from the perspective of size distribution and granular morphology.

Granular size induces the operation performance variation of aerobic granular sludge reactor, but the profound reasons are unrevealed. This study investigated the influence of granular size distribution on the reactor operation under salt stress. The effective nitrogen removal was achieved at ≤4% salinity, but declined at 6% salinity. The phenomenon was determined by the granular size fraction. The small granules (d=200-600μm) fraction was 77%-81% at ≤4% salinity, while only 57.32% at 6% salinity. That was positively correlated with nitrite reductase (NIR) activity significantly (p<0.01). Moreover, small granules exhibited smooth surface at ≤4% salinity. The efficient mass transfer area of granules was enlarged by the smooth surface, accelerating substrates mass transfer. Consequently, ammonia monooxygenase (AMO) activity was enhanced significantly (p<0.05). Ammonia-oxidation bacteria, Nitrosomonas and nitrite-reduction bacteria, Paracoccus were dominated in small granules at 4% salinity, while loss at 6% salinity. Overall, small granules with smooth surface favored the enrichment of nitrogen removal microbes via substrate transfer enhancement, and improved the activity of AMO and NIR. Thus, the favorable nitrogen removal performance of aerobic granular sludge reactor was achieved at ≤4% salinity.

Impact of carbon/nitrogen ratio on sequencing batch biofilm reactors initiated with different seed sludges for treating actual mariculture effluents.

The impact of carbon/nitrogen (C/N) ratio on sequencing batch biofilm reactor (SBBR) initiated with different seed sludges for treating actual mariculture effluent was explored. Increasing the C/N ratio significantly enhanced the nitrogen removal efficiency, achieving average removal efficiency of 95% for ammonia nitrogen and 73% for total nitrogen at ratio of 30, while the impact of seed sludge was minimal. High C/N ratio promoted the secretion of tightly bound extracellular polymeric substances (TB-EPS), which showed significant correlation with nitrogen removal. Interactions between bacteria and archaea were enhanced and conditionally rare or abundant taxa were the keystone taxa. High C/N ratio inhibited the relative abundance of ammonia-oxidizing archaea (Candidatus_Nitrosopumilus) and bacteria (Nitrosomonas), but promoted the heterotrophic nitrification-aerobic denitrification bacteria (Halomonas). The expression of nitrogen removal functional genes significantly correlated with functional genera. This study emphasized the crucial role of high C/N ratios in biological nitrogen removal from actual mariculture effluent.

Nitrogen removal from multi-electrolyte saline wastewater via mainstream anammox in warm climate conditions.

High salts concentrations in wastewater hinder its biological treatment. Recent research has investigated the inhibitory effect of salinity on the anammox process, mainly focusing on NaCl. Thus, the inhibition caused by multi-electrolytes salinity on freshwater anammox bacteria remains unclear. In this study, the anammox process was evaluated for the treatment of multi-electrolyte saline wastewater (NaCl, MgCl2, and CaCl2) during 684 days in three operational phases. In Phase 1, the anammox inoculum was successfully adapted from sidestream (232 mgN.L-1) to mainstream (60 mgN.L-1) conditions, with no damage to the reactor performance, at an hydraulic retention time of 1.4h. In Phase 2, salinity was gradually increased in the synthetic medium to adapt the freshwater anammox bacteria. The anammox bacteria tolerated a total salinity of 0.72wt% (in g.L-1: 4.7 NaCl, 2.0 MgCl2, and 0.6 CaCl2), achieving an 84.3±0.8% nitrogen removal efficiency. The presence of salts favored the Ca. Jettenia genus over Ca. Brocadia after long-term exposure to salinity. Finally, in Phase 3, anaerobically pre-treated saline wastewater (0.72wt%) was applied to the anammox reactor. The presence of residual organic matter (53 mgCOD.L-1; COD/N of 0.86) resulted in partial deviation of the metabolic pathway from anammox to, especially, nitrite heterotrophic denitrification, resulting in the accumulation of ammonia-N in the effluent. Even so, the anammox process was predominant, being responsible for 83% of the nitrogen removal. The presence of both organic matter and salinity led to a shift in dominance from the Ca. Jettenia genus to Ca. Brocadia.

Effect of light intensity on nitrogen removal, enzymatic activity and metabolic pathway of algal-bacterial symbiosis in rotating biological contactor treating mariculture wastewater.

An algal-bacterial symbiosis (ABS) system was developed on a rotating biological contactor treating mariculture wastewater, and its nitrogen removal, enzymatic activity and metabolic pathways were investigated under different light intensities. The nitrogen removal efficiency increased when light intensities ranged from 20 to 80 μE/(m2·s) but declined under 100 μE/(m2·s). Higher enzymatic activities under 80 μE/(m2·s) facilitated nitrogen conversion, light utilization, ATP supply and photosynthesis. Reactive oxygen species accumulation activated antioxidant pathways under 20 and 100 μE/(m2·s). Functional bacteria including Sedimentitalea, Thauera and Dechloromonas as well as Chlorella sorokinian, Dunaliella, Pleurosira laevis and Microcystis were enriched under 80 μE/(m2·s). Abundant photosynthesis-related genes (petC, Lca3/4 and atpH/A) supported energy supply and electron transport. Conversely, lower proportions of IDH3, gltB, and acnA/B under 20 and 100 μE/(m2·s) hindered tricarboxylic acid cycle, reducing NADPH and energy production. These results enhance the understanding on the effect of light intensity on ABS system treating mariculture wastewater.

Coupling Thiosulfate-Driven denitrification and anammox to remove nitrogen from actual wastewater.

A coupled thiosulfate-driven denitrification and anammox (TDDA) process was established to remove nitrogen from wastewater. It was optimized in an up-flow anaerobic sludge blanket reactor using synthetic wastewater, and its reliability was then verified with actual wastewater. The results demonstrated that nitrate, nitrite, and ammonium could be synergistically removed, and the highest total nitrogen removal efficiency reached 97.8% at a loading of 1.39 kgN/(m3·d). Anammox bacteria, primarily Candidatus_Brocadia, were the main contributors to nitrogen removal, while sulfur-oxidizing bacteria such as Thiobacillus and Rhodanobacter played a supportive role. By optimizing substrate conditions to enhance the anammox process, the coupled system attained higher abundances of functional genes such as napA, nirS, hzs, soxXA, and soxYZ, along with the corresponding microbial species. The data suggested that microbial cross-feeding and self-adaptation strategies were key to efficient nitrogen removal by TDDA.

Effects of multiple temperature variations on nitrogen removal and microbial community structure in tidal flow constructed wetlands.

Tidal-flow constructed wetlands (TFCWs) provide distinct advantages for nitrogen removal by enhancing microbial activity through dynamic water level fluctuations. However, effects of temperature on nitrogen transformation processes and microbial community dynamics in TFCWs remain unclear. We analyzed the effects of TFCWs on nitrogen transformation and microbial community structure under different temperature conditions (23, 16, 12, and 8 °C) through 140 days of temperature-controlled experiments. The nitrogen removal efficiency was considerably enhanced at 23 °C, with transformation rates for ammonia nitrogen (NH4+-N) and total nitrogen (TN) reaching 9.28 ± 0.06 g/m³/day and 8.35 ± 0.08 g/m³/day, respectively. Conversely, at 8 °C, the nitrogen removal efficiency declined, with NH4+-N and TN transformation rates decreasing to 7.38 ± 0.05 g/m³/day and 6.78 ± 0.05 g/m³/day, respectively. Temperature markedly influenced the microbial diversity and community structure, as evidenced by the considerably higher Shannon diversity indices for bacterial communities at 23 °C (5.12 ± 0.21) compared with those at 8 °C (4.52 ± 0.40). Positive microbial interactions were more prevalent at lower temperatures (12 and 8 °C), leading to stronger symbiotic relationships, although the network complexity diminished. The microbial community composition of taxa such as Firmicutes, Proteobacteria, and Thaumarchaeota exhibited greater resilience at lower temperatures. Changes in dissolved oxygen levels also drove changes in bacterial and archaeal communities. These findings underscore the pivotal role of temperature in regulating ecological function and nitrogen removal efficiency of TFCWs and highlight the importance of accounting for temperature variations in the design and management of wastewater treatment systems.

Rapid start-up and metabolic evolution of partial denitrification/anammox process by hydroxylamine stimulation: Nitrogen removal performance, biofilm characteristics and microbial community.

Enhanced nitrogen removal by hydroxylamine (NH2OH) on anammox-related process recently received attention. This study investigated the impact of NH2OH on the partial-denitrification/anammox (PDA) biosystem. Results show that NH2OH (≤10mg N/L) immediately induced nitrite accumulation and provided sufficient NO2- to anammox, achieving a 18.1±4.3% increase of nitrogen removal efficiency compared to the absence of NH2OH. Long-term exposure to NH2OH accelerated the functional microbial community transformation to PDA. Thauera was highly enriched (6.1%→26.9%) along with Candidatus Brocadia increased in the biofilms, which mainly favor the coupling process of nitrate reduction and anammox. Although the migration mechanism of anammox and denitrifier revealed by CLSM-FISH alleviates the adverse effects of NH2OH, the anammox was inhibited when NH2OH exceeding 15mg N/L through destroying the inner reduction of NO2-. These results suggested appropriate NH2OH addition favors the synergy between denitrifying and anammox bacteria, providing a promising option for wastewater treatment.

Application of the nitrogen-to-argon ratio to understand nitrogen transformation pathways in landfills under in-situ stabilization.

The ratio of nitrogen (N2) to argon (Ar) in landfill gas was compared to the atmospheric gas ratio to quantify the balance between N2 generating (anaerobic ammonium oxidation, denitrification) and N2 consuming (nitrogen fixation) processes on three landfills undergoing in-situ stabilization. In the aerated landfills, as much as 22% of the extracted N2 could be explained by net denitrification, with coexisting aerobic and anaerobic domains fostering nitrification-dependent denitrification. Nitrogen fixation was also occasionally observed. Removal of nitrogen via the gas phase exceeded nitrogen removed via the leachate by up to a factor of 33. Contrastingly, the anaerobic landfill under leachate recirculation showed a net reduction of N2 in relation to Ar, indicating nitrogen fixation as the dominant mechanism, equivalent up to 28% of the nitrogen in the extracted landfill gas. The balance between denitrification and nitrogen fixation in the aerated sites varied seasonally, likely caused by increased evapotranspiration in the summer, allowing greater air intrusion through the cover soil, resulting in higher NO3- and NO2- availability for denitrification and anammox. No such variability was observed for the landfill under liquid recirculation. The nitrogen transforming microbial community comprised of species responsible for nitrification, ammonification, denitrification, and anammox, indicating all processes may coexist. The findings show aeration supports nitrogen removal through the gas phase, but also suggest that nitrogen fixation adds nitrogen to the waste body in anaerobic domains. This could delay reaching environmental compliance criteria for leachate nitrogen, both for in-situ treatment by aeration and by leachate recirculation.

The start-up of red blood cell-like anammox granular sludge based on substrate volume flux

Enhancing substrate transfer is crucial for promoting the nitrogen removal rate of Anammox granular sludge, as the shape of the granules significantly influences substrate diffusion. In this study, inspired by biomimicry principles, we propose a novel red blood cell-like granular sludge to increase the contact area and improve substrate transfer. The startup and operation of Anammox granular sludge in low-strength ammonia nitrogen wastewater were successfully achieved through an increase in substrate volume flux (SVF). As the SVF increased from 2.38 to 7.43L²/(g VSS·h), specific Anammox activity (SAA) rose from 0.138 to 0.483gN/(g VSS·d). The Anammox granular sludge system achieved a total nitrogen removal efficiency of 85.70% and a nitrogen removal rate (NRR) of 0.792kgN/(m³·d) after 126 days. Notably, red blood cell-like granular sludge was observed by day 116. The rise in SVF induced a granule structure more conducive to substrate transport, improving the granules' efficiency in substrate uptake and thereby enhancing their SAA. High-throughput pyrosequencing revealed that the dominant bacteria using the SVF-enhancing strategy remained anaerobic ammonium oxidizing bacteria (AnAOB) (29.53%-29.89%), with an increased diversity of species, including Candidatus Kuenenia, Candidatus Anammoxoglobus, and Candidatus Brocadia.

Nitrogen removal in vertical flow constructed wetlands: The influence of recirculation and partial saturation

Nitrogen removal in vertical flow constructed wetlands: The influence of recirculation and partial saturation

Molecular ecological insights into the synergistic response mechanism of nitrogen transformation, electron flow and antibiotic resistance genes in aerobic activated sludge systems driven by sulfamethoxazole and/or trimethoprim stresses

The prevalence of antibiotics poses a serious challenge to biological nitrogen removal in wastewater. In this study, the effects of sulfamethoxazole and/or trimethoprim (15 mg/L∼30 mg/L) on treatment performance, nitrogen transformation and antibiotic resistance genes (ARGs) were investigated in aerobic activated sludge systems to elucidate the metabolic mechanism under high antibiotic stress. 15 mg/L single antibiotic stress improved total nitrogen removal performance due to the persistence of nitrifiers and enrichment of denitrifiers, with an optimum removal efficiency of 96.5%. Up-regulation of all denitrifying genes, coupled with enhanced electron transfer of Complex II and III, contributed to the emergence of aerobic denitrification. The increased expression of antioxidant genes also alleviated intracellular pressure. Whereas combined antibiotic stress induced the significant down-regulation of denitrifying bacteria and genes (nirKS and nosZ), and suppressed the electron supply for denitrification by restraining genes related to Complex Ⅰ and energy supply by tricarboxylic acid cycle, driving the collapse of activated sludge system, with ammonia and total nitrogen removal efficiencies dropping to below 40% and 20%, respectively. The dominant genera in system changed from TM7a to Thiothrix and Sphaerotilus with increasing antibiotic concentration and type. Moreover, antibiotic stress promoted a slight enrichment of ARGs, especially those encoding efflux mechanisms. Cooperative relationships (> 93%) dominated among ARGs, and Klebsiella was identified as the crucial host. ARGs regulating antibiotic efflux were more likely to be co-expressed with functional genes. These results may provide a theoretical basis for establishing promising strategies to mitigate antibiotic-caused process deterioration.

Long-term monitoring reveals improvements in nitrogen removal and energy efficiency with MABR upgrade at full scale

Long-term monitoring reveals improvements in nitrogen removal and energy efficiency with MABR upgrade at full scale

Effects of Ammonia loading and oxygen flux on nitrogen removal in a PNA-coupled MABR

Effects of Ammonia loading and oxygen flux on nitrogen removal in a PNA-coupled MABR

Insight into enhanced enrichment and nitrogen removal performance of Anammox bacteria with novel biochar/tourmaline polyurethane sponge modified biocarrier

A novel biochar/tourmaline polyurethane sponge modified biocarrier (BTP) could enhance Anammox bacteria (AnAOB) enrichment and nitrogen removal performance. With higher hydrophilicity and specific surface area, BTP significantly improved total inorganic nitrogen (TIN) removal efficiency to 80 ± 2 %, compared to unmodified biocarrier of 67 ± 3 % when influent TIN reached 633.9 ± 22.0 mg/L. BTP stimulated the upregulation of amino acid synthases genes abundance and improved protein secretion in extracellular polymer substances (EPS). Moreover, significant increases were found in heme concentration, specific anammox activity and hydrazine dehydrogenase of AnAOB with BTP compared to unmodified biocarrier. Extracellular electron transfer pathway of AnAOB was improved by BTP via upregulating cytochrome C and ferredoxin synthesis. Candidatus Brocadia was the main genus in Anammox biofilm, with relative abundance of 20.1 % and 27.6 % in the control and BTP, respectively, which explained the improvement of nitrogen removal performance

Simultaneous degradation of roxithromycin and nitrogen removal by Acinetobacter pittii TR1: Performances, pathways, and mechanisms.

Pharmaceutical and aquaculture wastewater contains not only antibiotics but also high concentrations of nitrogen, but few studies have been conducted on bacteria that target this complex pollution for degradation. A novel heterotrophic nitrifying aerobic denitrifying (HN-AD) strain Acinetobacter pittii TR1 isolated from soil. When the C/N ratio was 20, the strain could degrade 50mg/L roxithromycin (ROX) and the nitrogen removal rate was 96.06% with no accumulation of nitrite. The nitrogen metabolism pathway of strain TR1 was conjectured by NH4+-N → (NH2OH) → NO2--N → NO3--N → NO2--N → (NO → N2O → N2). Nitrogen balance analysis showed that 50.43% of the initial total nitrogen (TN) was converted to gaseous nitrogen and 45.39% was assimilated by TR1. Lower concentrations of ROX affected the activity of key enzymes, and the expression of the denitrifying genes hao, napA, nirK, norZ, and nosZ were significantly up-regulated, with the gene expression of nirK being up-regulated by 15.9-fold. Cleavage of desosamine, demethylation, and phosphorylation were the main biotransformation pathways of ROX. This work offers fresh insights into the metabolic processes of HN-AD bacteria under antibiotic stress, as well as evidence supporting strain TR1 in the treatment of wastewater with nitrogen and ROX.

Effective denitrification from landfill leachate using magnetic PVA/CMC/DE carrier immobilized microorganisms.

Ammonia nitrogen (NH4+-N) discharge has caused eutrophication of water bodies and harm to humans and organisms. In this work, polyvinyl alcohol (PVA), sodium carboxymethyl cellulose (CMC), diatomite (DE), and Fe3O4 were used to prepare magnetic immobilized carriers by encapsulating microorganisms for the treatment of NH4+-N wastewater. The response surface methodology was used to explore the optimal ratio of the immobilized carriers. The obtained optimal raw material ratio was 99.10%. The obtained carriers are spherical (4-5mm in diameter) with a rich honeycombed pore structure. The magnetic carrier improves the ammonia oxidation activity, and the carrier achieved 99.0% of NH4+-N and 86.7% of total nitrogen (TN) removal rates from the simulated wastewater (NH4+-N concentration: 300mg/L) through nitrification and denitrification under aerobic conditions. Upon applied for a 60days' treatment of landfill leachate (NH4+-N concentration of 300mg/L), the daily removal rates for NH4+-N and TN reached 93.7% and 78.3%, respectively. The analysis of the microbial community showed that the abundances of heterotrophic nitrifying-aerobic denitrifying bacteria including Enterobacter, Pseudomonas, and Bacillus increased with prolonging treatment days, which accelerated nitrification and denitrification, consequently promoting the nitrogen removal effect.