Biological Nitrogen Removal Systems Research Articles

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.

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.

Effect of thermal shock and sustained heat treatment on mainstream partial nitrification and microbial community in sequencing batch reactors.

In order to develop a promising means of achieving mainstream short-cut nitrification, this study evaluated the effect of thermal shock on nitrite accumulation using intermittent offline and continuous inline heat treatment of biomass in sequencing batch reactors (SBRs). The SBRs fed with municipal wastewater were operated at a solid retention time of 7 days and nitrogen loading rate of 0.04 gN/L·d to 0.08 gN/L·d without the application of pre-treatment. Contrary to literature studies that showed suppression of nitrite-oxidizing bacteria at temperature 60 to 80 °C, nitrite accumulation was achieved temporarily when 20% of the biomass was heated for 2 h at 47 °C, as well as in continuously heated SBRs at 37 °C and 42 °C. The continuously heated reactors at 37 °C and 42 °C produced a maximum nitrite accumulation ratio (NAR) of 0.59 and 0.79, respectively, whereas the intermittent offline heating at 47 °C-2 h produced a NAR of 0.37. Although nitrite accumulation was stable only for 10-12 days in all heated reactors, this study demonstrates the achievement of mainstream partial nitrification (PN) at lower temperature (42 °C) than that reported in literature and also highlights the potential for achieving PN by implementing heat treatment of a portion of the return activated sludge (RAS) in biological nitrogen removal (BNR) systems. During the time when full nitrification was achieved, Nitrospira was more dominant than Nitrosomonas in all reactors at ratios of 1.4:1, 2.4:1, 2.4:1, and 3.7:1 for the control SBR (22 °C), 47 °C -2 h offline heating SBR, 37 °C SBR, and 42 °C SBR, respectively, suggesting that it may have played a role as a comammox bacteria capable of degrading ammonia to nitrates at elevated temperature.

Successive exposure to traditional disinfectants and quaternary ammonium compounds enhances resistance genes expression and co-selection in biofilm-based partial nitrification-anammox systems

Quaternary ammonium compounds (QACs) are recommended disinfectants with surfactant properties, surpassing triclosan (TCS) and chloroxylenol (PCMX). Given the transition from traditional disinfectants, it is essential to investigate their impacts on biological nitrogen removal systems and the fate of resistance genes (RGs). In this study, three biofilm-based partial nitrification-anammox (PN/A) systems were established. A reactor named PD was successively exposed to 1 mg/L PCMX and 3 mg/L dioctadecyldimethylammonium chloride (DODMAC, a common QACs). A reactor named TD was successively exposed to 1 mg/L TCS and 3 mg/L DODMAC. A reactor named CD served as a control with only 3 mg/L DODMAC exposure. Results indicated that the total nitrogen removal performance of CD deteriorated markedly with DODMAC exposure compared to that of PD and TD. This phenomenon correlated closely with variations in RGs and their co-selection patterns. Pre-exposure to PCMX or TCS increased the abundance of RGs in the extracellular DNA of the PN/A biofilm, but reduced RGs abundances in the extracellular DNA of water. The tolerance of the PN/A system to successive exposure to the two disinfectants may be strengthened through co-selection of QACs RGs (qacEdelta1–01, qacEdelta1–02, qacH-01 and qacH-02) and mobile genetic elements (intI1 and tnpA-04). Furthermore, potential hosts of RGs are crucial for maintaining PN/A performance. Accumulation of extracellular polymeric substances, reactive oxygen species, and lactate dehydrogenase plays vital roles in the accumulation and transmission of RGs within the PN/A system.

Microbial community and gene dynamics response to high concentrations of gadolinium and sulfamethoxazole in biological nitrogen removal system

The impact of high antibiotic and heavy metal pollution levels on biological nitrogen removal in wastewater treatment plants (WWTPs) remains poorly understood, posing a global concern regarding the issue spread of antibiotic resistance induced by these contaminants. Herein, we investigated the effects of gadolinium (Gd) and sulfamethoxazole (SMX), commonly found in medical wastewater, on biological nitrogen removal systems and microbial characteristics, and the fate of antibiotic resistance genes (ARGs), metal resistance genes (MRGs), and mobile genetic elements (MGEs). Our findings indicated that high SMX and Gd(III) concentrations adversely affected nitrification and denitrification, with Gd(III) exerting a strong inhibitory effect on microbial activity. Metagenomic analysis revealed that high SMX and Gd(III) concentrations could reduce microbial diversity, with Thauera and Pseudomonas emerging as dominant genera across all samples. While the relative abundance of most ARGs decreased under single Gd(III) stress, MRGs increased, and nitrification functional genes were inhibited. Conversely, combined SMX and Gd(III) pollution increased the relative abundance of intl1. Correlation analysis revealed that most genera could host ARGs and MRGs, indicating co-selection and competition between these resistance genes. However, most denitrifying functional genes exhibited a positive correlation with MRGs. Overall, our study provides novel insights into the impact of high concentrations of antibiotics and heavy metal pollution in WWTPs, and laying the groundwork for the spread and proliferation of resistance genes under combined SMX and Gd pollution.

The response of nitrifying activated sludge to chlorophenols: Insights from metabolism and redox homeostasis

The specialized wastewater treatment plants for the chemical industry are rapidly developed in China and many other countries. But there is a common bottleneck in that the toxic pollutants in chemical wastewater often cause shock impacts on biological nitrogen removal systems, which directly affects the stability and cost of operation. As the research on nitrification inhibition characteristics is not sufficient till now, there is a great lack of theoretical guidance on the control of the inhibition. This study investigated the response of nitrifying activated sludge to chlorophenols (CPs) inhibition in terms of metabolism disorder and oxidative stress. At the initial stage of reaction (i.e., 1 h), reactive oxygen species (ROS)-induced membrane damage which might account for declining nitrification performance. Simultaneously excessive extracellular polymeric substances (EPS) were secreted to alleviate oxidative stress injury and protected microorganisms to some extent. In particular tyrosine-like substances in LB-EPS with a Fmax increase of 242.30% were confirmed to efficiently resist phenols inhibition. Thus, as the inhibition proceeded, metabolism disorder replaced oxidative stress as the main cause of nitrification inhibition. The affected metabolic processes include weakened enzyme catalysis, restricted electron transport and lessened energy generation. At 4 h, nitrifying production of sludge amended with 5 mg/L chlorophenols was 89.27 ± 9.51%–98.15 ± 9.60% lower than blank, the inhibition could be attributed to comprehensively affected metabolism. The structural equation modeling indicated that phenols restricted nitrification enzymes and bacterial electron transport efficiency which was critical to nitrification performance. Moreover, the lessened energy generation weakens enzyme activity to further suppress nitrification. These findings enriched our knowledge of nitrifiers’ responses to CPs inhibition and provided the basis for addressing nitrification inhibition.

Stress response mechanism of wastewater biological nitrogen removal systems to environmentally realistic concentrations of tire wear particles: Contribution of leachable additives

The study quantified the biological nitrogen removal performance, microbial metabolism, microbial community structure, and antioxidant system in a sequencing batch reactor under long-term exposure to 0.1 and 1 mg/L tire wear particles (TWPs), and determined the contribution of leachable additives to the biotoxicity of TWPs. The results showed that long-term exposure to 0.1 and 1 mg/L TWPs inhibited both the nitrification and denitrification processes, reducing ammonia nitrogen (NH4+-N) and total nitrogen (TN) removal efficiency. The TWP leachate (TWPL) primarily contributed to the denitrification inhibition by TWPs, potentially due to the high concentration of zinc ions in the leachable additive. Furthermore, both TWP and TWPL inhibit nitrogen conversion, with TWP inhibiting the generation and transfer of electrons, while TWPL only negatively affects the electron transfer process. This study presents novel insights into the impact of TWPs on biological nitrogen removal, underscoring its broader implications for the geochemical nitrogen cycle.

Metabolic inhibitor-free assessment of the heterotrophic ammonia-oxidizing activity in activated sludge

When the contributions of three ammonia-oxidizing pathways (heterotrophic or autotrophic aerobic ammonia oxidization, and anammox) to wastewater biological nitrogen removal systems was compared by determining their ammonia-oxidizing activities, the key question is how to accurately determine the potential heterotrophic aerobic ammonia-oxidizing (PHAe) activity when the potential autotrophic aerobic ammonia-oxidizing (PAAe) activity (by ammonia-oxidizing bacteria (AOB) or archaea, or complete ammonia oxidization bacteria) also contributes to ammonia oxidization in PHAe activity assay medium. Using a AOB species and three heterotrophic AOB species as inocula, we demonstrated the feasibility of PHAe activity evaluation in the absence of a metabolic inhibitor, i.e., by subtracting the PAAe activity determined in PAAe activity assay medium from a combination of PAAe and PHAe activity determined in PHAe activity assay medium. Binary organic carbon sources (i.e., glucose and acetate) were included in the PHAe activity assay medium to fulfill the carbon requirements of most heterotrophic AOB genera. Higher ammonia-oxidizing activity in AOB biomass than heterotrophic AOB biomass (35.6 vs. 2.6–10.0 mg NH4+–N g−1 MLSS h−1) provides the remarkable advantages of autotrophic aerobic ammonia oxidization in biological nitrogen removal systems. Ammonia removal in three full-scale biological nitrogen removal systems for sewage treatment was predominantly mediated by PAAe activity (1.9–3.3 vs. 0.0–0.3 mg NH4+–N g1 MLSS h−1).

Insight into response characteristics and inhibition mechanisms of anammox granular sludge to polyethylene terephthalate microplastics exposure

Currently, in-depth understanding of response characteristics and mechanisms of anammox process under microplastics (MPs) stress remains quite limited. This study investigated the influence of 0.1–1.0 g/L polyethylene terephthalate (PET) on anammox granular sludge (AnGS). Compared with the control, 0.1–0.2 g/L PET did not significantly affect the anammox efficiency, while the anammox activity decreased by 16.2% at 1.0 g/L PET. Integrity coefficient and transmission electron microscopy analysis demonstrated that the strength and structural stability of the AnGS became weaken following exposure to 1.0 g/L PET. With the PET increasing, the abundance of anammox genera and genes related to energy metabolism and cofactors and vitamins metabolism decreased. The reactive oxygen species generated in the interaction between microbial cells and PET resulting in cellular oxidative stress was responsible for inhibiting anammox. These findings give novel insights into the anammox behavior in biological nitrogen removal systems treating PET-loaded nitrogenous wastewater.

New insights into exogenous N-acyl-homoserine lactone manipulation in biological nitrogen removal system against ZnO nanoparticle shock

The effects and mechanisms of three N-acyl-homoserine lactones (AHLs) (C4-HSL, C6-HSL, and C10-HSL) on responses of biological nitrogen removal (BNR) systems to zinc oxide nanoparticle (NP) shock were investigated. All three AHLs improved the NP-impaired ammonia oxidation rates by up to 50.0 % but inhibited the denitrification process via regulating nitrogen metabolism-related enzyme activities. C4-HSL accelerated the catalase activity by 13.2 %, while C6-HSL and C10-HSL promoted the superoxide dismutase activity by 26.6 % and 18.4 %, respectively, to reduce reactive oxygen species levels. Besides, the enhancements of tryptophan protein and humic acid levels in tightly-bound extracellular polymeric substance by AHLs were vital for NP toxicity attenuation. The metabonomic analysis demonstrated that all three AHLs up-regulated the levels of lipid- and antioxidation-related metabolites to advance the system’s resistance to NP shock. The “dual character” of AHLs emphasized the concernment of legitimately employing AHLs to alleviate NP stress for BNR systems.

Effect of peracetic acid solution on a nitrifying culture: Kinetics, inhibition, cellular and transcriptional responses

Peracetic acid (PAA) has been widely used as a disinfectant in many industries. However, information related to the potential inhibitory effect of PAA solutions (PAA and H2O2) on biological wastewater treatment processes is very limited. The work reported here assessed the effect of PAA and H2O2 solutions on nitrification kinetics and inhibition, cellular level responses and gene expression of a suspended-growth nitrifying culture. The initial ammonia removal and nitrate production rates significantly decreased at 1/0.14 to 3/0.42 mg/L PAA/H2O2. H2O2 up to 3 mg/L did not impact nitrification, cell viability or related respiratory activities; thus, the impact of the PAA solution is attributed to PAA alone or in some combination with H2O2. Nitrification inhibition by PAA was predominantly related to enzyme inhibition, rather than to loss of cell viability and/or cell lysis. PAA and H2O2 negatively affected Nitrosomonas but resulted in Nitrosospira enrichment. Most nitrogen metabolism-related genes (e.g., hydroxylamine oxidoreductase and nitrite oxidoreductase genes) as well as oxidase genes (e.g., cytochrome c oxidase, catalase-peroxidase, and peroxidase genes) were upregulated in PAA- and H2O2-amended cultures. Major ATPase genes were downregulated while ATP synthase genes upregulated under the effect of PAA and/or H2O2. Upregulation of ATP-dependent protease genes indicates protein damage predominantly caused by PAA rather than H2O2. The transcriptional level of genes related to cell division and DNA repair did not show a particular pattern; thus, cell division functionality and DNA integrity were not significantly affected by PAA or H2O2. The results of this study have significant implications in the design and operation of effective biological nitrogen removal systems for the treatment of PAA-bearing wastewater.

Organic carbon determines nitrous oxide consumption activity of clade I and II nosZ bacteria: Genomic and biokinetic insights

Harnessing nitrous oxide (N2O)-reducing bacteria is a promising strategy to reduce the N2O footprint of engineered systems. Applying a preferred organic carbon source as an electron donor accelerates N2O consumption by these bacteria. However, their N2O consumption potential and activity when fed different organic carbon species remain unclear. Here, we systematically compared the effects of various organic carbon sources on the activity of N2O-reducing bacteria via investigation of their biokinetic properties and genomic potentials. Five organic carbon sources—acetate, succinate, glycerol, ethanol, and methanol—were fed to four N2O-reducing bacteria harboring either clade I or clade II nosZ gene. Respirometric analyses were performed with four N2O-reducing bacterial strains, identifying distinct shifts in DO- and N2O-consumption biokinetics in response to the different feeding schemes. Regardless of the N2O-reducing bacteria, higher N2O consumption rates, accompanied by higher biomass yields, were obtained with acetate and succinate. The biomass yield (15.45 ± 1.07 mg-biomass mmol-N2O−1) of Azospira sp. strain I13 (clade II nosZ) observed under acetate-fed condition was significantly higher than those of Paracoccus denitrificans and Pseudomonas stutzeri, exhibiting greater metabolic efficiency. However, the spectrum of the organic carbon species utilizable to Azospira sp. strain I13 was limited, as demonstrated by the highly variable N2O consumption rates observed with different substrates. The potential to metabolize the supplemented carbon sources was investigated by genomic analysis, the results of which corroborated the N2O consumption biokinetics results. Moreover, electron donor selection had a substantial impact on how N2O consumption activities were recovered after oxygen exposure. Collectively, our findings highlight the importance of choosing appropriate electron donor additives for increasing the N2O sink capability of biological nitrogen removal systems.

Recent advancements in the biological treatment of high strength ammonia wastewater

The estimated global population growth of 81 million people per year, combined with increased rates of urbanization and associated industrial processes, result in volumes of high strength ammonia wastewater that cannot be treated in a cost-effective or sustainable manner using the floc-based conventional activated sludge approach of nitrification and denitrification. Biofilm and aerobic granular sludge technologies have shown promise to significantly improve the performance of biological nitrogen removal systems treating high strength wastewater. This is partly due to enhanced biomass retention and their ability to sustain diverse microbial populations with juxtaposing growth requirements. Recent research has also demonstrated the value of hybrid systems with heterogeneous bioaggregates to mitigate biofilm and granule instability during long-term operation. In the context of high strength ammonia wastewater treatment, conventional nitrification-denitrification is hampered by high energy costs and greenhouse gas emissions. Anammox-based processes such as partial nitritation-anammox and partial denitrification-anammox represent more cost-effective and sustainable methods of removing reactive nitrogen from wastewater. There is also growing interest in the use of photosynthetic bacteria for ammonia recovery from high strength waste streams, such that nitrogen can be captured and concentrated in its reactive form and recycled into high value products. The purpose of this review is to explore recent advancements and emerging approaches related to high strength ammonia wastewater treatment.

Nitrification and denitrification in recirculating aquaculture systems: the processes and players

AbstractOne of the major challenges in any sustainable aquaculture production systems is the accumulation of nitrogenous waste such as ammonia and its biological nitrification products viz nitrite and nitrate. Considering the bio‐security issues, amelioration of these wastes without water exchange can be accomplished only by way of establishing in situ nitrification and denitrification through biofilters/bioreactors activated with nitrifying/denitrifying bioaugmentors. In such systems, coexistence of aerobic denitrifiers, anaerobic ammonia oxidizers (Anammox) and complete ammonia oxidizers (Comammox) together with the autotrophic nitrifiers enhance the coupled nitrification–denitrification. This promotes total nitrogen removal without external carbon supplements or additional anerobic compartment in the system. Various recirculating aquaculture systems (RAS) comprise diverse nitrifying community in biofilters/bioreactors thereby imparting distinctive nitrogen conversion in the system. Meanwhile, the structure and population dynamics of the nitrifying/denitrifying consortia are influenced by the environmental factors forming the decisive factors of the success of the processes. Accordingly, understanding the complexity of nitrifying/denitrifying community composition turns out to be a requirement to facilitate its improvised performance. In this context, the review addresses different biological nitrogen removal systems, significance of nitrification–denitrification in RAS, genetic diversity of the key players in RAS, methods of analysis of their community structure, current application and prospects of the processes in RAS.

Identification of primary effecters of N2O emissions from full-scale biological nitrogen removal systems using random forest approach

Wastewater treatment plants (WWTPs) have long been recognized as point sources of N2O, a potent greenhouse gas and ozone-depleting agent. Multiple mechanisms, both biotic and abiotic, have been suggested to be responsible for N2O production from WWTPs, with basis on extrapolation from laboratory results and statistical analyses of metadata collected from operational full-scale plants. In this study, random forest (RF) analysis, a machine-learning approach for feature selection from highly multivariate datasets, was adopted to investigate N2O production mechanism in activated sludge tanks of WWTPs from a novel perspective. Standardized measurements of N2O effluxes coupled with exhaustive metadata collection were performed at activated sludge tanks of three biological nitrogen removal WWTPs at different times of the year. The multivariate datasets were used as inputs for RF analyses. Computation of the permutation variable importance measures returned biomass-normalized dissolved inorganic carbon concentration (DIC·VSS−1) and specific ammonia oxidation activity (sOURAOB) as the most influential parameters determining N2O emissions from the aerated zones (or phases) of activated sludge bioreactors. For the anoxic tanks, dissolved-organic-carbon-to-NO2−/NO3− ratio (DOC·(NO2−-N + NO3−-N)−1) was singled out as the most influential. These data analysis results clearly indicate disparate mechanisms for N2O generation in the oxic and anoxic activated sludge bioreactors, and provide evidences against significant contributions of N2O carryover across different zones or phases or niche-specific microbial reactions, with aerobic NH3/NH4+ oxidation to NO2− and anoxic denitrification predominantly responsible from aerated and anoxic zones or phases of activated sludge bioreactors, respectively.

Metatranscriptomic analysis of adaptive response of anammox bacteria Candidatus ‘Kuenenia stuttgartiensis’ to Zn(II) exposure

Zn(II) is frequently detected in biological nitrogen removal systems treating high-strength wastewater (e.g., landfill leachate), yet the cellular defense strategies of anammox bacteria against Zn(II) cytotoxicity is largely unknown. To uncover survival mechanisms under Zn(II) stress, responses of enriched anammox bacteria Candidatus 'Kuenenia stuttgartiensis' under exposure of various levels of Zn (II) were investigated through metatranscriptomic sequencing. Although increasing Zn(II) levels (50, 100 and 150mg/L) resulted in decreasing anammox activities (86.1±0.8%, 66.1±1.4% and 43.9±1.5% of the control, respectively), the viable cells in anammox sludge remained stable. Candidatus 'K. stuttgartiensis' possesses a complex network of regulatory systems to confer cells with the ability against Zn(II) toxicity, including functions related to substrate degradation, Zn(II) efflux, chelation, DNA repair, protein degradation, protein synthesis and signal transduction processes. Particularly, in order to maintain Zn(II) homeostasis, Candidatus 'K. stuttgartiensis' upregulated genes encoding RND efflux family (czcA, czcB, czcC, kustd1923 and kuste2279) for exporting Zn(II) actively. These heavy metal exporting genes could act as "sentinel genes" to detect the initial stage of Zn(II) inhibition on anammox bacteria, which might be beneficial to develop a diagnostic approach to predict the risk of operational failure when Zn(II) shock occurs.

Effects of Exogenous N-Acyl-Homoserine Lactone as Signal Molecule on Nitrosomonas Europaea under ZnO Nanoparticle Stress.

Despite the adverse effects of emerging ZnO nanoparticles (nano-ZnO) on wastewater biological nitrogen removal (BNR) systems being widely documented, strategies for mitigating nanoparticle (NP) toxicity impacts on nitrogen removal have not been adequately addressed. Herein, N-acyl-homoserine lactone (AHL)-based quorum sensing (QS) was investigated for its effects against nano-ZnO toxicity to a model nitrifier, Nitrosomonas europaea. The results indicated that AHL-attenuated nano-ZnO toxicity, which was inversely correlated with the increasing dosage of AHL from 0.01 to 1 µM. At 0.01 µM, AHL notably enhanced the tolerance of N. europaea cells to nano-ZnO stress, and the inhibited cell proliferation, membrane integrity, ammonia oxidation rate, ammonia monooxygenase activity and amoA gene expression significantly increased by 18.2 ± 2.1, 2.4 ± 0.9, 58.7 ± 7.1, 32.3 ± 1.7, and 7.3 ± 5.9%, respectively, after 6 h of incubation. However, increasing the AHL dosage compromised the QS-mediated effects and even aggravated the NPs’ toxicity effects. Moreover, AHLs, at all tested concentrations, significantly increased superoxide dismutase activity, indicating the potential of QS regulations to enhance cellular anti-oxidative stress capacities when facing NP invasion. These results provide novel insights into the development of QS regulation strategies to reduce the impact of nanotoxicity on BNR systems.

Abiotic Nitrous Oxide (N2O) Production Is Strongly pH Dependent, but Contributes Little to Overall N2O Emissions in Biological Nitrogen Removal Systems.

Hydroxylamine (NH2OH) and nitrite (NO2-), intermediates during the nitritation process, can engage in chemical (abiotic) reactions that lead to nitrous oxide (N2O) generation. Here, we quantify the kinetics and stoichiometry of the relevant abiotic reactions in a series of batch tests under different and relevant conditions, including pH, absence/presence of oxygen, and reactant concentrations. The highest N2O production rates were measured from NH2OH reaction with HNO2, followed by HNO2 reduction by Fe2+, NH2OH oxidation by Fe3+, and finally NH2OH disproportionation plus oxidation by O2. Compared to other examined factors, pH had the strongest effect on N2O formation rates. Acidic pH enhanced N2O production from the reaction of NH2OH with HNO2 indicating that HNO2 instead of NO2- was the reactant. In departure from previous studies, we estimate that abiotic N2O production contributes little (< 3% of total N2O production) to total N2O emissions in typical nitritation reactor systems between pH 6.5 and 8. Abiotic contributions would only become important at acidic pH (≤ 5). In consideration of pH effects on both abiotic and biotic N2O production pathways, circumneutral pH set-points are suggested to minimize overall N2O emissions from nitritation systems.

Structural and Functional Interrogation of Selected Biological Nitrogen Removal Systems in the United States, Denmark, and Singapore Using Shotgun Metagenomics.

Conventional biological nitrogen removal (BNR), comprised of nitrification and denitrification, is traditionally employed in wastewater treatment plants (WWTPs) to prevent eutrophication in receiving water bodies. More recently, the combination of selective ammonia to nitrite oxidation (nitritation) and autotrophic anaerobic ammonia oxidation (anammox), collectively termed deammonification, has also emerged as a possible energy- and cost-effective BNR alternative. Herein, we analyzed microbial diversity and functional potential within 13 BNR processes in the United States, Denmark, and Singapore operated with varying reactor configuration, design, and operational parameters. Using next-generation sequencing and metagenomics, gene-coding regions were aligned against a custom protein database expanded to include all published aerobic ammonia oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB), anaerobic ammonia oxidizing bacteria (AMX), and complete ammonia oxidizing bacteria (CMX). Overall contributions of these N-cycle bacteria to the total functional potential of each reactor was determined, as well as that of several organisms associated with denitrification and/or structural integrity of microbial aggregates (biofilm or granules). The potential for these engineered processes to foster a broad spectrum of microbial catabolic, anabolic, and carbon assimilation transformations was elucidated. Seeded sidestream DEMON® deammonification systems and single-stage nitritation-anammox moving bed biofilm reactors (MBBRs) and a mainstream Cleargreen reactor designed to enrich in AOB and AMX showed lower enrichment in AMX functionality than an enriched two-stage nitritation-anammox MBBR system treating mainstream wastewater. Conventional BNR systems in Singapore and the United States had distinct metagenomes, especially relating to AOB. A hydrocyclone process designed to recycle biomass granules for mainstream BNR contained almost identical structural and functional characteristics in the overflow, underflow, and inflow of mixed liquor (ALT) rather than the expected selective enrichment of specific nitrifying or AMX organisms. Inoculum used to seed a sidestream deammonification process unexpectedly contained <10% of total coding regions assigned to AMX. These results suggest the operating conditions of engineered bioprocesses shape the resident microbial structure and function far more than the bioprocess configuration itself. We also highlight the advantage of a systems- and metagenomics-based interrogation of both the microbial structure and potential function therein over targeting of individual populations or specific genes.

Comparison of N2O Emissions and Gene Abundances between Wastewater Nitrogen Removal Systems.

Biological nitrogen removal (BNR) systems are increasingly used in the United States in both centralized wastewater treatment plants (WWTPs) and decentralized advanced onsite wastewater treatment systems (OWTS) to reduce N discharged in wastewater effluent. However, the potential for BNR systems to be sources of nitrous oxide (NO), a potent greenhouse gas, needs to be evaluated to assess their environmental impact. We quantified and compared NO emissions from BNR systems at a WWTP (Field's Point, Providence, RI) and three types of advanced OWTS (Orenco Advantex AX 20, SeptiTech Series D, and Bio-Microbics MicroFAST) in nine Rhode Island residences ( = 3 per type) using cavity ring-down spectroscopy. We also used quantitative polymerase chain reaction to determine the abundance of genes from nitrifying () and denitrifying () microorganisms that may be producing NO in these systems. Nitrous oxide fluxes ranged from -4 × 10 to 3 × 10 µmol NO m s and in general followed the order: centralized WWTP > Advantex > SeptiTech > FAST. In contrast, when NO emissions were normalized by population served and area of treatment tanks, all systems had overlapping ranges. In general, the emissions of NO accounted for a small fraction (<1%) of N removed. There was no significant relationship between the abundance of or genes and NO emissions. This preliminary analysis highlights the need to evaluate NO emissions from wastewater systems as a wider range of technologies are adopted. A better understanding of the mechanisms of NO emissions will also allow us to better manage systems to minimize emissions.