Enhancing NosZ Activity to Reduce N2O Emissions from Biological Wastewater Treatment Systems

Enhancing NosZ Activity to Reduce N2O Emissions from Biological Wastewater Treatment Systems | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Enhancing NosZ Activity to Reduce N 2 O Emissions from Biological Wastewater Treatment Systems View ORCID Profile Xueyang Zhou , View ORCID Profile Bharat Manna , View ORCID Profile Boyu Lyu , View ORCID Profile Naresh Singhal doi: https://doi.org/10.1101/2024.09.22.614384 Xueyang Zhou 1 Department of Civil and Environmental Engineering, University of Auckland , Auckland 1010, New Zealand 2 Water Research Centre, University of Auckland , Auckland 1010, New Zealand Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Xueyang Zhou Bharat Manna 1 Department of Civil and Environmental Engineering, University of Auckland , Auckland 1010, New Zealand 2 Water Research Centre, University of Auckland , Auckland 1010, New Zealand Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Bharat Manna Boyu Lyu 1 Department of Civil and Environmental Engineering, University of Auckland , Auckland 1010, New Zealand 2 Water Research Centre, University of Auckland , Auckland 1010, New Zealand Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Boyu Lyu Naresh Singhal 1 Department of Civil and Environmental Engineering, University of Auckland , Auckland 1010, New Zealand 2 Water Research Centre, University of Auckland , Auckland 1010, New Zealand Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Naresh Singhal For correspondence: n.singhal{at}auckland.ac.nz Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Nitrous oxide (N 2 O) emissions from wastewater treatment plants, with a warming potential 298 times that of CO 2 , pose a significant challenge to lowering their carbon footprint. Current mitigation strategies focus on limiting N 2 O formation during nitrification and denitrification but overlook microbial reduction mechanisms. This study examines the potential for enhancing nitrous oxide reductase (NosZ) activity to reduce N 2 O to N 2 . We hypothesize that strategic oxygen manipulation can enhance N 2 O destruction by continuous NosZ expression and enable NosZ activation in microorganisms with superior NosZ capabilities. We assess microbial community function and metabolic regulation using metagenomics and metaproteomics to clarify the effect of intermittent aeration regimes on N 2 O emission. Intermittent aeration with periodic anoxic exposure significantly reduced N 2 O emissions with 71% nitrogen removal by enhancing the metabolic activity of Hyphomicrobium . NosZ activity increased by 4- to 6.5-fold after system adaptation to oxygen modulations, compared to continuous oxic-anoxic cycling without the anoxic phase. The latter resulted in increased N 2 O emissions due to suppressed NosZ activity and higher N 2 O production from Methylobacillus , which uses nitric oxide as an alternative electron acceptor. Our finding that strategic oxygen manipulation can energize N 2 O destruction lays the foundation for developing next-generation wastewater treatment technologies for mitigating N 2 O emissions. INTRODUCTION Nitrous oxide (N 2 O) emissions from biological nitrogen removal processes, which have a global warming potential 298 times greater than carbon dioxide, pose a significant challenge for wastewater treatment plants aiming to achieve carbon neutrality 1 – 3 . Despite extensive research, current N 2 O mitigation strategies remain constrained by a production-centric focus, overlooking the transformative potential of microbial reduction mechanisms 4 – 6 . In contrast to managing N 2 O production during nitrification and denitrification 7 – 10 , the true potential for mitigation lies in the biological destruction pathway—specifically, the reduction of N 2 O to N 2 by nitrous oxide reductase (NosZ), the only enzyme capable of complete N 2 O elimination 11 – 15 . This research challenges conventional strategies 1 , 4 , 16 – 19 by exploring how targeted deoxygenation can unlock the latent N 2 O destruction capabilities of microbial communities 20 . Recent studies reveal a nuanced interplay between oxygen availability and N 2 O reduction, highlighting the metabolic flexibility of denitrifying microorganisms 21 , 22 . Historical oxygen manipulation studies show mixed results 23 , with some reducing N 2 O emissions 4 , 5 , 24 , 25 and others increasing them 26 , 27 . These inconsistencies underscore a critical knowledge gap in understanding the mechanisms linking oxygen variation to N 2 O dynamics 4 , 5 , 28 – 30 . We hypothesize that strategic oxygen manipulation can enhance N 2 O destruction through three key mechanisms: (i) maintaining continuous NosZ expression, (ii) enabling NosZ activation, and (iii) selectively enhancing microorganisms with superior NosZ capabilities. This approach shifts from traditional emission management to active microbial-driven N 2 O elimination. Our study investigates microbial community function and metabolic regulation under controlled intermittent aeration regimes. We aim to identify key microbial contributors, their enzymatic pathways, and the regulatory mechanisms governing N 2 O production and reduction under varying oxygen conditions. By bridging fundamental microbial mechanisms with practical applications, we aim to provide an evidence-based framework for developing next-generation wastewater treatment technologies for effectively mitigating N 2 O emissions from biological wastewater treatment processes. MATERIALS AND METHODS Bioreactor Operation Activated sludge used in this study was sourced from the Māngere Wastewater Treatment Plant in Auckland, New Zealand. Three identical cylindrical acrylic bioreactors, each with a 1 L working volume, were operated simultaneously under varying aeration conditions at 20⏢. The same activated sludge was used in triplicate to ensure biological consistency. Each 1 L reactor contained 2.75 g/L of mixed liquor-suspended solids and was supplemented with 3.84 g/L of NaHCO3 as an inorganic carbon source, along with 1 mL of a trace element solution (Table S1). Over a 48-hour period, 50 mL of concentrated artificial wastewater (Table S2) was continuously fed into each reactor at an equal rate using a syringe pump, while magnetic stirring ensured uniform mixing. The bioreactors were operated under three aeration modes, aimed at suppressing denitrification, inducing N 2 O production, or promoting N 2 O reduction, respectively. Each mode was tested at two oxygen levels, resulting in six conditions: constant aerobic (CA) with steady oxygen levels of 2 and 8 mg/L ( Figure 1a, 1b ); continuous perturbed (CP) with cyclic oxygen variations of 0-2 and 0-8 mg/L without intermittent anoxic stage ( Figure 1c, 1d ); and, intermittent perturbed (IP) with cyclic oxygen variations that include an anoxic stage ( Figure 1e, 1f ). Download figure Open in new tab Figure 1. Dissolved oxygen (DO) concentration profiles under six aeration conditions: CA2, CA8, CP2, CP8, IP2, and IP8. Each plot highlights distinct modes of oxygen availability, showcasing constant aerobic (CA), continuous perturbed (CP), and intermittent perturbed (IP) modes at low (2 mg/L) and high (8 mg/L) DO levels. N 2 O Emissions Gaseous N 2 O emissions were quantified using gas chromatography (Shimadzu GC-BID/FID 2010 Plus), and dissolved N 2 O concentrations were monitored with online N 2 O sensors (Unisense, Denmark). Detailed analytical methods, including sensor calibration, gas sampling, and data processing procedures, are provided in Supplementary Materials S1.3. In-situ N 2 O reductase (N 2 OR) Activity In-situ N 2 OR activity was assessed by introducing a saturated N 2 O solution into the bioreactor and monitoring the consumption of dissolved N 2 O using an online sensor (Unisense, Denmark). The N 2 O consumption rate was determined by analyzing the time-dependent decrease in dissolved N 2 O concentrations. Metagenomics DNA was extracted from sludge samples using the DNeasy PowerSoil Kit (Qiagen, Germany) following the manufacturer’s protocol. Samples were collected at the start of the experiment (six biological replicates) for nitrogen transformer identification and at the 48-hour mark under dissolved oxygen (DO) 2 and DO8 conditions to construct the metaproteomics library. The extracted DNA underwent Illumina sequencing on a HiSeq platform. Details on sample preparation, sequencing procedures, and bioinformatics analysis using SqueezeMeta 31 are provided in Supplementary Materials S1.4. Metaproteomics Protein extraction and analysis were conducted for sludge samples collected under different aeration conditions. Biological replicates included five from the start of the experiment, three from 48-hour DO2, and two from 48-hour DO8 conditions. Proteins were extracted, purified, digested, and analyzed via nano LC-MS/MS using a TripleTOF 6600 mass spectrometer. Protein identification was conducted against a database established by metagenomics results of the samples using MetaProteomeAnalyzer version 3.4 32 . Full details of the experimental protocol are available in Supplementary Materials S1.5. RESULTS Nitrogen Removal Under Perturbed Aeration Under CA conditions at both tested DO concentrations (CA2 and CA8), nitrogen removal was minimal (<3%), indicating the predominance of aerobic metabolic pathways (Figure S1, S2). CP conditions (CP2 and CP8) facilitated limited denitrification, resulting in approximately 8% conversion of nitrogen to gaseous forms (Figure S3, S4). In contrast, IP conditions (IP2 and IP8) enabled significant nitrogen removal (∼71%), driven by enhanced denitrification through strategic oxygen availability patterns (Figure S5, S6). Role of Oxygen Modulation in N 2 O Dynamics To understand the role of oxygen modulation in N 2 O dynamics, we analyzed dissolved and gaseous N 2 O emissions under three aeration modes—CA, CP, and IP. The N 2 O emissions show distinct trends in dynamics ( Figure 2 ). IP showed lower dissolved N 2 O accumulation and stabilized gaseous emissions than CP, which was comparable to the N 2 O emissions under CA. N 2 O transformation dynamics exhibited three characteristic phases ( Figure 2 ): an initial accumulation phase (Phase I), a depletion phase (Phase II), and lastly a stabilization phase (Phase III). During Phase I, the dissolved N 2 O concentrations increased across all aeration modes and showed significant variation among the observed rates for different conditions at 95% confidence (Table S3). The dissolved N 2 O accumulation rates during the initial phase were significantly lower under IP (0.034 mg/L/h for IP2 and 0.007 mg/L/h for IP8) compared to CP (0.064 mg/L/h for CP2 and 0.089 mg/L/h for CP8). Gaseous N 2 O emissions mirrored these trends, with substantially lower N 2 O-N emitted in the first 8 hours under IP (5.99 mg N and 5.08 mg N under IP2 and IP8, respectively) than CP (9.32 mg N and 12.07 mg N under CP2 and CP8, respectively). This reduction in initial accumulation suggests that anoxic intervals inherent to IP strategies effectively limit N 2 O production pathways. During Phase II, the N 2 O emissions decreased, in particular for CP2, CP8, and IP2. The depletion rates under IP2 (-0.022 mg/L/h) were comparable to CP (-0.023 mg/L/h for CP2 and -0.039 mg/L/h for CP8); however, IP consistently showed lower gaseous emissions throughout this phase (0.73 to 0.21 mg N/hour under IP2 versus 1.94 to 0.29 mg N/hour under CP2). This trend continued until stabilization was achieved. Phase III data show that while the dissolved N 2 O concentrations were lowest under IP (0.11–0.26 mg/L for IP2 and 0.036–0.12 mg/L for IP8), gaseous emissions became negligible across all conditions, signaling an equilibrium between N 2 O production and reduction processes. Moreover, NosZ expression and activation, leading to higher N 2 OR activity and dissolved N 2 O consumption rates, were highest under IP (1.23 mg N 2 O-N/L/min and 4.19 mg N 2 O-N/L/min for IP2 and IP8, respectively) and showed 4- to 6.5-fold exceedance over CP conditions with the same oxygen range ( Figure 3 , Table S4). Download figure Open in new tab Figure 2. Gaseous and dissolved N 2 O concentration dynamics under six aeration conditions: CA2, CA8, CP2, CP8, IP2, and IP8. Gaseous N 2 O emissions were measured during 0–8 hours, 22–32 hours, and 46–48 hours, while dissolved N 2 O concentrations were continuously monitored over 48 hours. Download figure Open in new tab Figure 3. In situ N 2 O reductase (N 2 OR) activity, measured as the rate of dissolved N 2 O consumption after 48 hours of exposure to six aeration conditions. Results indicate enhanced N 2 OR activity under IP conditions compared to CA and CP modes. Microbial Contributors to N 2 O Emissions Metagenomic and metaproteomic analyses ( Figure 4 , Figure S7) identified the key organisms responsible for N 2 O dynamics and how aeration strategies influence their metabolic activities. Nitrosomonas is the dominant ammonia-oxidizing genus that catalyzes the conversion of ammonia to hydroxylamine and subsequently to nitrite, and Nitrospira is the primary nitrite-oxidizing genus for nitrite to nitrate oxidation ( Figure 4 ); both possess genes for partial denitrification pathways to convert nitrite to nitric oxide (NO) and N 2 O, potentially contributing to N 2 O emissions. Methylobacterium and Hyphomicrobium stand out as the two main denitrifying genera ( Figure 4 ) but possess different denitrification functional genes. Methylobacterium contains genes for NO and N 2 O production and lacks genes for N 2 O reduction, making it a potential source of N 2 O production. In contrast, Hyphomicrobium has a complete set of denitrification-related genes, which includes the nosZ gene, and could contribute to N 2 O reduction. Download figure Open in new tab Figure 4. Metagenomic insights into microbial community structure and functional differentiation of nitrogen-transforming genera. (a) Source microorganisms and their contribution to nitrogen transformation-related genes. (b) Functional gene differentiation among nitrogen-transforming genera. An enzymatic analysis showed significant differences in the abundances of denitrification enzymes under different aeration conditions (Figure S7). Nitric oxide reductase (NorB) and NosZ emerged as critical enzymes driving the N 2 O dynamics. NorB, responsible for N 2 O production, showed higher abundance under CP, compared to CA and IP, and its activity was primarily attributed to Methylobacterium , a genus within the class Betaproteobacteria ( Figure 5 ). Conversely, NosZ, which facilitates N 2 O reduction, was significantly higher under IP and is primarily associated with Hyphomicrobium within the class Alphaproteobacteria ( Figure 5 ). The differences in enzyme abundance under different aeration conditions mirror the observed N 2 O emissions. Since N 2 O producers and reducers tend to favor different aeration modes, oxygen availability under some aeration patterns could influence the interactions between N 2 O producers and reducers, resulting in dynamic changes in the dissolved N 2 O pool. Download figure Open in new tab Figure 5. Microbial sources of nitric oxide reductase (NorB) and nitrous oxide reductase (NosZ) identified through metaproteomic analysis. Contributions under CP and IP conditions illustrate distinct microbial drivers for N 2 O production and reduction. Mechanisms of N 2 O Metabolism A detailed metaproteome analysis revealed distinct adaptation strategies employed by Methylobacillus and Hyphomicrobium under different aeration patterns. Increasing the oxygen level from 2 to 8 mg/L did not significantly affect these strategies. Methylobacillus Adaptations Under CP conditions, Methylobacillus exhibited higher expression of cytochrome c oxidase cbb3-type and NorB, indicating that N 2 O formation occurs as a metabolic byproduct under limited oxygen availability ( Figure 6a ). This adaptation was supported by stabilized NO concentrations of approximately 14 μM (Figure S8), enabling sustained electron transport chain function through NO utilization. The absence of NosZ in Methylobacillus resulted in its inability to reduce the N 2 O it produced. Two-way ANOVA analysis (Figure S9) showed that aeration mode significantly influenced Methylobacterium metabolism (p = 0.003), while oxygen level had an insignificant effect (p = 0.74). Overall enzyme expression in Methylobacterium declined under IP conditions compared to CA and CP conditions, contributing to the suppression of N 2 O production (Figure S10). Download figure Open in new tab Figure 6. Metabolic regulatory mechanisms of N 2 O-related processes in (a) Methylobacillus under CP conditions, characterized by upregulation of nitric oxide reductase and cytochrome c oxidase cbb3-type, and (b) Hyphomicrobium under IP conditions, showing enhanced electron generation and upregulation of nitrate reductase and nitrous oxide reductase. Hyphomicrobium Adaptations Hyphomicrobium exhibited elevated enzyme expression ability under IP conditions (p = 0.008) (Figure S11), contrasting with the metabolic response patterns observed in Methylobacterium . This indicates that Hyphomicrobium possesses a distinct adaptive mechanism to redox fluctuations, including a strong potential for reducing N 2 O (Figure S12). Under IP conditions, Hyphomicrobium demonstrated comprehensive metabolic reprogramming ( Figure 6b ). Enhanced expression of carbon dissimilation enzymes included upregulation of methanol dehydrogenase (Mdh), methylene-tetrahydromethanopterin dehydrogenase (MtdB), and NADH dehydrogenase. Upregulation of nitrate reductase (NarGHJI) and NosZ enhanced energy production and N 2 O reduction capabilities under low-oxygen conditions. A key regulatory adaptation in Hyphomicrobium involved upregulating the CRP/FNR family transcriptional regulator, specifically the dissimilatory nitrate respiration regulator (DNR) ( Figure 6b ). This regulator responded to elevated NO concentrations (∼21 μM) (Figure S8), coordinating terminal enzyme expression patterns. Integrating these adaptive mechanisms enabled Hyphomicrobium to effectively utilize NO as a signal, optimizing anaerobic respiration and N 2 O reduction under IP conditions. DISCUSSION Our investigation into strategic oxygen manipulation in wastewater treatment systems has revealed complex mechanisms governing N 2 O transformation. Detailed analyses of N 2 O dynamics, enzyme regulation, and metabolic adaptations provide strong evidence supporting our hypothesis that controlled oxygen availability can enhance community-wide N 2 O destruction through three interconnected mechanisms. Continuous NosZ Expression Contrary to conventional assumptions about oxygen sensitivity, we demonstrated that Hyphomicrobium maintains continuous NosZ expression under specific intermittent aeration (IP conditions). Stable enzyme levels were exhibited after the system adapted to the aerobic-anoxic transition, suggesting metabolic resilience and adaptability essential for effective N 2 O mitigation. This finding aligns with observations from other systems, including mixed microbial communities (e.g., soil microorganisms 33 and permeable sediments 34 ) and specific pure cultures (e.g., Azospira sp . 22 , Thauera sp . 35 , and Pseudomonas stutzeri 36 ), where NosZ expression occurs due to relaxed regulation of respiratory genes by oxygen and metabolic flexibility to exploit periodic supplies of electron acceptors under fluctuating oxygen conditions 37 – 39 . Enhanced NosZ Activation Limiting oxygen by introducing an anoxic phase in the IP cycles significantly enhanced NosZ activation, resulting in markedly higher N 2 O reduction rates under IP conditions compared to CA and CP conditions. This suggests a sophisticated regulatory mechanism in Hyphomicrobium , similar to the resilience seen in Azospira sp ., which demonstrates rapid recovery of NosZ activity after oxygen exposure 22 , 40 . Additionally, evidence suggests that denitrification proteins expressed under oxic conditions remain functional, further supporting this adaptability 34 . This tolerance ensures sustained carbon metabolism and continuous electron flow for energy production, regardless of oxygen availability 5 , 41 , 42 , highlighting NosZ’s critical role in maintaining robust N 2 O reduction under fluctuating environmental conditions 43 , 44 . Selective Enhancement of NosZ-Containing Microorganisms The oxygen manipulation strategy selectively enhanced microorganisms with superior NosZ capabilities, particularly Hyphomicrobium , which leveraged its complete denitrification pathway and metabolic flexibility to sustain robust N 2 O reduction even under fluctuating oxygen conditions 39 , 45 . In contrast, Methylobacillus , which operates with an incomplete denitrification pathway, relied on cbb3-type cytochrome c oxidase and NorB to maximize the utilization of electron acceptors 46 . However, this adaptation led to increased N 2 O production, highlighting the drawbacks of incomplete denitrifiers in dynamic environments 47 , 48 . Ecological Dynamics and Community-Wide Impact The contrasting responses of these microorganisms emphasize the importance of targeting complete denitrifiers in optimizing N 2 O mitigation strategies. The shared utilization of denitrification intermediates among microbial communities suggests an ecological advantage in enhancing nosZ-containing species 45 . Given that the potential for N 2 O reduction often surpasses its production capacity 20 , fostering these organisms can amplify their collective impact, creating a synergistic effect that reduces N 2 O emissions and contributes to the overall efficiency of nitrogen cycling within the microbial ecosystem 49 . Implications for N 2 O Mitigation Strategies Implementing strategies solely targeting N 2 O production may be suboptimal 50 . While stable oxygen conditions (CA) effectively suppress N 2 O production, they lack resilience to sudden oxygen fluctuations, often leading to abrupt N 2 O surges 9 , 51 , 52 . In contrast, specific IP conditions have been shown to offer superior ways of reducing N 2 O emissions 4 , 5 , 24 . Our study provides significant evidence that enhancing the N 2 O reduction capacity of nosZ-containing microorganisms plays a critical role in buffering against N 2 O accumulation, reinforcing the effectiveness of IP strategies. These findings underscore the importance of designing mitigation approaches that address both N 2 O production and reduction mechanisms 53 , 54 . Targeted enhancement of N 2 O destruction represents a promising pathway to optimize mitigation efforts and maintain stable system performance under variable operational conditions. This research establishes a comprehensive mechanistic framework for controlling N 2 O emissions in biological nitrogen removal systems, demonstrating the effectiveness of strategic oxygen manipulation in promoting NosZ activity for N 2 O reduction. Associated content Supporting information Detailed materials and methods, the performance of biosystems, N 2 O accumulation dynamics, N 2 O consumption rates, the abundance of denitrification-related enzymes, NO concentrations, and enzyme expression analysis (PDF) Author information Corresponding authors *Phone: +64 9 923 4512; fax: +64 9 373 7462; e-mail: n.singhal{at}auckland.ac.nz . Funding sources The study was supported by the Marsden Fund Council from Government funding, managed by Royal Society Te Apārangi, and a Smart Ideas grant from the Endeavour Fund managed by the Ministry for Business, Innovation & Employment Hīkina Whakatutuki. Acknowledgments We thank Nikki Freed for the assistance with metagenomic sequencing. We thank Martin Middleditch and George Guo for their assistance with metaproteomics analysis. We also thank Watercare Services Limited for providing the activated sludge culture from the Māngere Wastewater Treatment Plant. The authors acknowledge assistance from the New Zealand eScience Infrastructure (NeSI) high-performance computing facilities and the Centre for eResearch at the University of Auckland. Footnotes The manuscript has been revised to better describe the experimental phenomena and elucidate the mechanism. REFERENCES (1). ↵ Duan , H. ; Zhao , Y. ; Koch , K. ; Wells , G. F. ; Weißbach , M. ; Yuan , Z. ; Ye , L. Recovery of nitrous oxide from wastewater treatment: Current status and perspectives . ACS EST Water 2021 , 1 ( 2 ), 240 – 250 . doi: 10.1021/acsestwater.0c00140 . OpenUrl CrossRef (2). Ribera-Guardia , A. ; Bosch , L. ; Corominas , L. ; Pijuan , M. Nitrous oxide and methane emissions from a plug-flow full-scale bioreactor and assessment of its carbon footprint . 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