N2O Reductase Activity Research Articles

Effects of exogenous thermophilic bacteria and ripening agent on greenhouse gas emissions, enzyme activity and microbial community during straw composting

This research examined the impact of exogenous thermophilic bacteria and ripening agents on greenhouse gas (GHG) emission, enzyme activity, and microbial community during composting. The use of ripening agents alone resulted in a 30.9 % reduction in CO2 emissions, while the use of ripening agents and thermophilic bacteria resulted in a 50.8 % reduction in N2O emissions. Pearson’s analysis showed that organic matter and nitrate nitrogen were the key parameters affecting GHG emissions. There was an inverse correlation between CO2 and CH4 releases and methane monooxygenase α subunit and N2O reductase activity (P<0.05). Additionally, N2O emissions were positively related to β-1, 4-N-acetylglucosaminidase, and ammonia monooxygenase activity (P<0.05). Deinococcota, Chloroflexi, and Bacteroidota are closely related to CO2 and N2O emissions. Overall, adding thermophilic bacteria represents an effective strategy to mitigate GHG emissions during composting.

Nitrous oxide respiration in acidophilic methanotrophs

Aerobic methanotrophic bacteria are considered strict aerobes but are often highly abundant in hypoxic and even anoxic environments. Despite possessing denitrification genes, it remains to be verified whether denitrification contributes to their growth. Here, we show that acidophilic methanotrophs can respire nitrous oxide (N2O) and grow anaerobically on diverse non-methane substrates, including methanol, C-C substrates, and hydrogen. We study two strains that possess N2O reductase genes: Methylocella tundrae T4 and Methylacidiphilum caldifontis IT6. We show that N2O respiration supports growth of Methylacidiphilum caldifontis at an extremely acidic pH of 2.0, exceeding the known physiological pH limits for microbial N2O consumption. Methylocella tundrae simultaneously consumes N2O and CH4 in suboxic conditions, indicating robustness of its N2O reductase activity in the presence of O2. Furthermore, in O2-limiting conditions, the amount of CH4 oxidized per O2 reduced increases when N2O is added, indicating that Methylocella tundrae can direct more O2 towards methane monooxygenase. Thus, our results demonstrate that some methanotrophs can respire N2O independently or simultaneously with O2, which may facilitate their growth and survival in dynamic environments. Such metabolic capability enables these bacteria to simultaneously reduce the release of the key greenhouse gases CO2, CH4, and N2O.

Effect of n-hexadecanoic acid on N2O emissions from vegetable soil and its synergism with Pseudomonas stutzeri NRCB010

Recently, it has been recommended to utilize specific plant root metabolites to decrease nitrous oxide (N2O) emissions from agricultural soil. However, only a limited number of plant root metabolites have been shown to decrease soil N2O emissions, and their combined effects with plant growth-promoting rhizobacteria, as well as the mechanisms involved in mitigating N2O emissions, remain to be explored. This study investigated the impact and microbial mechanisms of three tomato root exudate metabolites, namely, methyl hexadecanoate, methyl stearate, and n-hexadecanoic acid, on N2O emissions. In a pure culture experiment, n-hexadecanoic acid significantly enhanced Pseudomonas stutzeri NRCB010 auxin production, nitrogen removal, N2O reduction capacity, and N2O reductase activity. Methyl stearate significantly promoted NRCB010 N removal and the N2O reduction capacity. In soil microcosm and greenhouse pot experiments, n-hexadecanoic acid decreased N2O cumulative emissions by 34.3 % and 46.6 %, respectively, and further decreases were observed when combined with NRCB010. Additionally, n-hexadecanoic acid, both independently and in combination with NRCB010, significantly promoted tomato growth. N-hexadecanoic acid, alone or with NRCB010, also altered soil microbial communities and nitrogen-cycle functional gene abundance. Structural equation models revealed that n-hexadecanoic acid, either alone or with NRCB010, decreased N2O cumulative emissions by modifying the environmental conditions to be more conducive to N2O mitigation (e.g., higher soil pH and organic matter content), inhibiting N2O production (through a decrease in ammonia-oxidizing bacteria and nirK gene copy numbers), and promoting N2O reduction (by increasing nosZII gene copy numbers). Our results support the potential use of n-hexadecanoic acid in enhancing crop productivity and mitigating N2O emissions in agricultural soils.

Exposure of sulfur-driven autotrophic denitrification to hydroxylamine/hydrazine: Underlying mechanisms and implications for promoting partial denitrification and N2O recovery

Hydroxylamine (NH2OH) and hydrazine (N2H4) are crucial intermediates in biological nitrogen removal processes, but their potential effects on the nitrogen-related metabolic pathway during the sulfur-driven autotrophic denitrification (SDAD) process remain unclear and were thus explored in this work. The results of dedicated batch tests indicated that the accumulation ratio of NO2− was positively correlated with the NH2OH concentration, reaching 86.2 ± 2.6% at the NH2OH concentration of 2.0 mg-N/L, while it was barely affected by the N2H4 concentration. Furthermore, the joint inhibition of NH2OH and free nitrous acid led to the substantial accumulation of N2O (up to 32.9 ± 1.3%) during the NO2− reduction phase. In contrast, N2H4 exhibited great potential for facilitating N2O accumulation in both the NO3− reduction and NO2− reduction phases with an accumulation ratio of 13.0 ± 0.3% and 27.5 ± 1.5%, respectively, at the N2H4 concentration of 2.0 mg-N/L. Total enzyme activity analyses revealed that the presence of NH2OH inhibited the activity expression of all denitrifying enzymes, with the downstream enzymes (such as NO2− reductase and N2O reductase) being more susceptible to NH2OH. The addition of N2H4 did not cause a significant decrease in the activity of NO3− reductase and NO2− reductase, but notably curbed the activity of N2O reductase. Based on the responses of the SDAD-related nitrogen metabolic pathway to NH2OH/N2H4, this work provided effective technical means for i) the promotion of NO2− accumulation which might enable the coupling of SDAD with Anammox and ii) the efficient recovery of N2O via the SDAD process.

Evaluating the inhibitory effects of Nitrobenzene short-term stress on denitrification performance: Electron behaviors, bacterial and fungal community

Denitrifying system is a feasible way to remove nitro-aromatic compounds (NACs) in wastewater. However, the toxicity and mechanisms of NACs to denitrification remain unknown. This study investigated effects of nitrobenzene (NB, a typical NAC) on denitrification in short term. Results showed that NB in 10–50 mg/L groups decreased NO3−-N removal efficiency by 9%–24%, but increased nitrous oxide (N2O) generation by 6–17fold. Mechanistic research indicated that NB could deteriorate electron behaviors and disturbed enzyme activities of microbial metabolism and denitrification, leading to a decline in denitrification performance. Structural equation modeling revealed that N2O reductase activity was the core factor in predicting denitrification performance at exposure of NB, with the indirect effects of NADH and electron transport system activity. High-throughput sequencing analysis demonstrated that NB had made an alteration on both bacterial and fungal community structure, as well as their interactions.

Arbuscular mycorrhizal fungi drive soil nitrogen transformation under wheat varieties with different nitrogen utilization efficiencies

Arbuscular mycorrhizal fungi (AMF) can play important roles in plant nitrogen (N) uptake and use. However, it is still not well explored how AMF interact with host plants representing different N use types and thereby affect soil N transformation. A pot experiment was conducted to test the effects of AMF on N mineralization, nitrification, denitrification and nitrous oxide (N2O) reduction in soils under wheat varieties with high and low pre-anthesis N utilization efficiency (also called grain-specific N efficiency). Our results showed that AMF can increase N mineralization potential, with a greater effect for varieties with high N utilization efficiency. AMF also increased nitrification potential, ammonium oxidation potential, but did not nitrite oxidation potential, suggesting that AMF only act on the first nitrification step (ammonium oxidation). AMF increased NO3− but decreased NH4+ concentrations irrespective of the N use type of host plant. Moreover, AMF significantly increased denitrifying enzyme activity of the varieties with low N utilization efficiency, but decreased it for the varieties with high N utilization efficiency. N2O emission during soil incubation was in tandem with denitrification. Finally, AMF significantly increased N2O reductase activity irrespective of the N utilization efficiency of the host plant. The findings suggest that an interaction between the N utilization efficiency of wheat plants and AMF can modulate the N uptake of the host plant, and also regulate soil N transformation; a mechanism which may greatly affect the sustainability of wheat cropping systems.

Hysteretic response of N2O reductase activity to soil pH variations after application of lime to an acidic agricultural soil

Hysteretic response of N2O reductase activity to soil pH variations after application of lime to an acidic agricultural soil

Nitric oxide and Nitrous oxide accumulation, oxygen production during nitrite denitrification in an anaerobic/anoxic sequencing batch reactor: exploring characteristics and mechanism.

Nitrite denitrification has received increasing attention due to its high efficiency, low energy consumption, and sludge yield. However, the nitric oxide (NO) and nitrous oxide (N2O) which are harmful to the environment, microorganisms, and humans are produced in this process. In order to mitigate NO and N2O production, the biological mechanisms of NO and N2O accumulation, as well as NO detoxification during nitrite denitrification in a sequencing batch reactor were studied. Results showed that the peak of NO accumulation increased from 0.29 [Formula: see text] 0.01 to 3.12 [Formula: see text] 0.34mg L-1 with the increase of carbon to nitrogen ratio (COD/N), which is caused by the sufficient electron donor supply for NO2--N reduction process at high COD/N. Furthermore, the result suggested that NO accumulation with no pH adjustment was 12 times higher than that with pH adjustment. It is related to the inhibition on NO reductase caused by the high free nitrous acid (FNA) and NO concentration with no pH adjustment. The pathways of NO detoxification included NO emission, reduction, and dismutation, and the more NO produced, the high proportion of NO dismutation pathway. Result showed that the maximum of oxygen production during NO dismutation reached to 1.39mg L-1. N2O accumulation was mainly associated with FNA and NO inhibition, COD/N. The peak of N2O accumulation presented a completely opposite trend at pH adjustment and no pH adjustment, it is because that the higher FNA and NO concentration at high COD/N without pH adjustment will inhibit the N2O reductase activity, resulting in the N2O reduction was hindered during nitrite denitrification.

Differential immediate and long-term effects of nitrogen input on denitrification N2O/(N2O+N2) ratio along a 0‒5.2 m soil profile.

High nitrogen (N) input to soil can cause higher nitrous oxide (N2O) emissions, that is, a higher N2O/(N2O+N2) ratio, through an inhibition of N2O reductase activity and/or a decrease in soil pH. We assumed that there were two mechanisms for the effects of N input on N2O emissions, immediate and long-term effect. The immediate effect (field applied fertilizer N) can be eliminated by decreasing the N input, but not the long-term effect (soil accumulated N caused by long-term fertilization). Therefore, it is important to separate these effects to mitigate N2O emissions. To this end, soil samples along a 0‒5.2 m profile were collected from a long-term N fertilization experiment field with two N application rates, that is, 600 kg N ha-1 year-1 (N600) and no fertilizer N input (N0). External N addition was conducted for each subsample in the laboratory incubation study to produce two additional treatments, which were denoted as N600+N and N0+N treatments. The results showed that the combined immediate and long-term effects led to an increase in the N2O/(N2O+N2) ratio by 6.8%. Approximately 32.6% and 67.4% of increase could be explained by the immediate and long-term effects of N input, respectively. Meanwhile, the long-term effects were significantly positively correlated to soil organic carbon (SOC). These results indicate that excessive N fertilizer input to the soil can lead to increased N2O emissions if the soil has a high SOC content. The long-term effect of N input on the N2O/(N2O+N2) ratio should be considered when predicting soil N2O emissions under global environmental change scenarios.

Long-term exposure to nano-TiO2 interferes with microbial metabolism and electron behavior to influence wastewater nitrogen removal and associated N2O emission

The extensive use of nano-TiO2 has caused concerns regarding their potential environmental risks. However, the stress responses and self-recovery potential of nitrogen removal and greenhouse gas N2O emissions after long-term nano-TiO2 exposure have seldom been addressed yet. This study explored the long-term effects of nano-TiO2 on biological nitrogen transformations in a sequencing batch reactor at four levels (1, 10, 25, and 50 mg/L), and the reactor's self-recovery potential was assessed. The results showed that nano-TiO2 exhibited a dose-dependent inhibitory effect on the removal efficiencies of ammonia nitrogen and total nitrogen, whereas N2O emissions unexpectedly increased. The promoted N2O emissions were probably due to the inhibition of denitrification processes, including the reduction of the denitrifying-related N2O reductase activity and the abundance of the denitrifying bacteria Flavobacterium. The inhibition of carbon source metabolism, the inefficient electron transfer efficiency, and the electronic competition between the denitrifying enzymes would be in charge of the deterioration of denitrification performance. After the withdrawal of nano-TiO2 from the influent, the nitrogen transformation efficiencies and the N2O emissions of activated sludge recovered entirely within 30 days, possibly attributed to the insensitive bacteria survival and the microbial community diversity. Overall, this study will promote the current understanding of the stress responses and the self-recovery potential of BNR systems to nanoparticle exposure.

Chronic effects of cerium dioxide nanoparticles on biological nitrogen removal and nitrous oxide emission: Insight into impact mechanism and performance recovery potential

The influence of cerium dioxide nanoparticles (CeO2 NPs) on biological nitrogen removal and associated nitrous oxide (N2O) emission has seldom been addressed yet. Herein, the chronic effect of CeO2 NPs on the nitrogen transformation processes during wastewater treatment and the impacted system's self-recovery potential after CeO2 NP stress removal were investigated. CeO2 NP of 10–50 mg/L induced significant declines of the ammonia nitrogen (NH4+-N) and the total nitrogen removal efficiencies, but triggered the nitrite accumulation and the N2O emission. The N2O reductase (NOS) activity was negatively correlated with the N2O emission level, and the inhibition of NOS activity under CeO2 NP stress was probably due to the depressions of the sludge denitrifiers' metabolic activities. The NH4+-N removal efficiency was successfully regained after the recovery period although the N2O emission level was still higher than the pre-exposure period, which was probably due to the residual CeO2 NPs inside the activated sludge.

The accumulation characteristics of NO and N2O in A/O-SBR for treating ammonium-rich organic wastewater

The emissions of NO and N2O during biological nitrogen removal harm the environment. In this study, a sequencing batch reactor (SBR) was used to treat ammonium-rich organic wastewater. The nitrogen transformation and characteristics of NO and N2O accumulation in typical cycles under different nitrification conditions were monitored, and the mechanisms of NO and N2O production in A/O-SBR were analyzed. The results showed that NO accumulated in the anoxic phase under a fixed concentration of organic carbon source during denitrification with nitrite as electron acceptor or nitrate as electron acceptor. NO production in the aerobic phase could be related to hydroxylamine oxidation and nitrite reduction caused by high ammonia nitrogen. The accumulation of N2O mainly occurred in the aerobic phase, and the cumulative concentration of N2O at the end of the four typical cycles was above 4.02 mg/L. N2O accumulation attributed to the inhibition of free nitrous acid (FNA) and NO, and electronic competition, which made N2O reductase (N2OR) activity insufficient, failing N2O reduction.

Long-term effects of acetylene on denitrifying N2O production: Biomass performance and microbial community

Maximizing production and use of N2O produced by the process of denitrification has emerged as a promising concept for energy recovery from nitrogen. Acetylene is well known as an inhibitor of N2O reductase activity, however, to date, few studies have utilized long-term cultivation with acetylene to achieve denitrifying N2O recovery. This work assessed the performance of N2O production and shifts in the denitrifying community in response to long-term exposure of acetylene during nitrite denitrification. Batch tests showed that long-term exposure to acetylene at high concentrations (volume fraction >40 %) resulted in high N2O accumulation, up to 98.6 %. High-throughput 16S rRNA gene analysis suggested that Saprospiraceae dominated the microbial community at the end of the cultivation period with its abundance increasing from the initial 3.2% to 42.2%. Metagenomic analysis revealed that after the long-term cultivation under high level acetylene, the relative abundance of nosZ in the community did not change significantly. Instead, the nosZⅡ-harboring bacteria became dominant. Batch tests in series demonstrated that N2O reduction could be restored rapidly upon removal of acetylene. This study describes an effective method for high yield production of N2O during nitrite denitrification that can be used to enhance energy recovery and improves our understanding of the long-term effect of acetylene on the denitrifying community.

Long-term effects of chlorothalonil on microbial denitrification and N2O emission in a tea field soil.

Pesticide chlorothalonil is widely applied in tea agroecosystem, potentially disturbing soil microbial-mediated nitrogen cycle. The underlying toxicity mechanism, however, is not well explored. Here, we investigated the long-term effects of chlorothalonil on soil microbial denitrification and N2O emission pattern in a tea field after 40days of exposure. Results showed that chlorothalonil inhibited denitrification process but remarkably promoted N2O emission by 380-830%. Chlorothalonil significantly inhibited N2O reductase activity but did not affected nosZ abundance. Our results further revealed that chlorothalonil influenced soil denitrification by directly suppressing microbial electron transport system activity, and decreasing electron donor nicotinamide adenine dinucleotide (NADH) and energy source adenosine triphosphate (ATP) levels. Additionally, chlorothalonil also downregulated denitrifying functional genes (narG, nirS, and norB) and declined the relative abundances of potential denitrifiers (i.e., Pseudomonas and Streptomyces). Stepwise regression andpath modeling suggested that nitrate reductase was the most significant factor in explaining denitrification rate under chlorothalonil applications. This study provides important information for revealing the chronic impacts of pesticide on tea soil denitrification and N2O emission on the basis of electron transport mechanism. Most significantly, N2O emission is underestimated in chlorothalonil-treated soils, which suggests that future estimations of N2O emission from agricultural lands should take account of pesticide dependency conditions.

N2O Reductase Activity of a [Cu4S] Cluster in the 4CuI Redox State Modulated by Hydrogen Bond Donors and Proton Relays in the Secondary Coordination Sphere

AbstractThe model complex [Cu4(μ4‐S)(dppa)4]2+ (1, dppa=μ2‐(Ph2P)2NH) has N2O reductase activity in methanol solvent, mediating 2 H+/2 e− reduction of N2O to N2+H2O in the presence of an exogenous electron donor (CoCp2). A stoichiometric product with two deprotonated dppa ligands was characterized, indicating a key role of second‐sphere N−H residues as proton donors during N2O reduction. The activity of 1 towards N2O was suppressed in solvents that are unable to provide hydrogen bonding to the second‐sphere N−H groups. Structural and computational data indicate that second‐sphere hydrogen bonding induces structural distortion of the [Cu4S] active site, accessing a strained geometry with enhanced reactivity due to localization of electron density along a dicopper edge site. The behavior of 1 mimics aspects of the CuZ catalytic site of nitrous oxide reductase: activity in the 4CuI:1S redox state, use of a second‐sphere proton donor, and reactivity dependence on both primary and secondary sphere effects.

Nitrogen Cycling in Soybean Rhizosphere: Sources and Sinks of Nitrous Oxide (N2O).

Nitrous oxide (N2O) is the third most important greenhouse gas after carbon dioxide and methane, and a prominent ozone-depleting substance. Agricultural soils are the primary anthropogenic source of N2O because of the constant increase in the use of industrial nitrogen (N) fertilizers. The soybean crop is grown on 6% of the world’s arable land, and its production is expected to increase rapidly in the future. In this review, we summarize the current knowledge on N-cycle in the rhizosphere of soybean plants, particularly sources and sinks of N2O. Soybean root nodules are the host of dinitrogen (N2)-fixing bacteria from the genus Bradyrhizobium. Nodule decomposition is the main source of N2O in soybean rhizosphere, where soil organisms mediate the nitrogen transformations that produce N2O. This N2O is either emitted into the atmosphere or further reduced to N2 by the bradyrhizobial N2O reductase (N2OR), encoded by the nos gene cluster. The dominance of nos– indigenous populations of soybean bradyrhizobia results in the emission of N2O into the atmosphere. Hence, inoculation with nos+ or nos++ (mutants with enhanced N2OR activity) bradyrhizobia has proved to be promising strategies to reduce N2O emission in the field. We discussed these strategies, the molecular mechanisms underlying them, and the future perspectives to develop better options for global mitigation of N2O emission from soils.

The coupling interaction of NO2- with NH4+ or NO3- as an important source of N2O emission from agricultural soil in the North China Plain.

NO2- plays a crucial role in regulating N2O formation from the soil, while how it affects the production of soil N2O is still not well understood. In this study, N2O and NO emissions from an agricultural field of the North China Plain (NCP) were comparatively investigated under five different fertilizer treatments (NH4+, NO3-, NO2-, NH4+ + NO2- and NO3- + NO2-). Additionally, soil NH4+, NO2- and NO3- concentrations and the abundance of functional genes associated with nitrogen cycling were also analyzed in the incubation experiment. The results showed that the N2O average fluxes from the complex treatments of NO2- + NO3- were 1.4-2.4 times the sum of those from the separate treatments of NO2- and NO3- whereas from the complex treatments of NO2- + NH4+ were a factor of 1-1.4 larger than those from the separate treatments of NO2- and NH4+, indicating the coupling interaction of NO2- with NH4+ or NO3- makes a remarkable contribution to N2O emission from the soil. Significant reduction of the activity of N2O reductase was found in the soil with the addition of NO2-, which favored the accumulation of N2O formed through nitrification of NH4+ and denitrification of NO2-, resulting in relatively high N2O emissions from the complex treatments. As the intermediate product of nitrification and denitrification, NO2- produced is also expected to interact with NH4+ or NO3- to promote N2O emission from the soil, especially during fertilization events when NO2- is easily accumulated due to the acceleration of the nitrification and denitrification processes.

Ecological and physiological implications of nitrogen oxide reduction pathways on greenhouse gas emissions in agroecosystems.

Microbial reductive pathways of nitrogen (N) oxides are highly relevant to net emissions of greenhouse gases (GHG) from agroecosystems. Several biotic and abiotic N-oxide reductive pathways influence the N budget and net GHG production in soil. This review summarizes the recent findings of N-oxide reduction pathways and their implications to GHG emissions in agroecosystems and proposes several mitigation strategies. Denitrification is the primary N-oxide reductive pathway that results in direct N2O emissions and fixed N losses, which add to the net carbon footprint. We highlight how dissimilatory nitrate reduction to ammonium (DNRA), an alternative N-oxide reduction pathway, may be used to reduce N2O production and N losses via denitrification. Implications of nosZ abundance and diversity and expressed N2O reductase activity to soil N2O emissions are reviewed with focus on the role of the N2O-reducers as an important N2O sink. Non-prokaryotic N2O sources, e.g. fungal denitrification, codenitrification and chemodenitrification, are also summarized to emphasize their potential significance as modulators of soil N2O emissions. Through the extensive review of these recent scientific advancements, this study posits opportunities for GHG mitigation through manipulation of microbial N-oxide reductive pathways in soil.

Denitrification as an N2O sink

The strong greenhouse gas nitrous oxide (N2O) can be emitted from wastewater treatment systems as a byproduct of ammonium oxidation and as the last intermediate in the stepwise reduction of nitrate to N2 by denitrifying organisms. A potential strategy to reduce N2O emissions would be to enhance the activity of N2O reductase (NOS) in the denitrifying microbial community. A survey of existing literature on denitrification in wastewater treatment systems showed that the N2O reducing capacity (VmaxN2O→N2) exceeded the capacity to produce N2O (VmaxNO3→N2O) by a factor of 2–10. This suggests that denitrification can be an effective sink for N2O, potentially scavenging a fraction of the N2O produced by ammonium oxidation or abiotic reactions. We conducted a series of incubation experiments with freshly sampled activated sludge from a wastewater treatment system in Oslo and found that the ratio α = VmaxN2O→N2/VmaxNO3→N2O fluctuated between 2 and 5 in samples taken at intervals over a period of 5 weeks. Adding a cocktail of carbon substrates resulted in increasing rates, but had no significant effect on α. Based on these results – complemented with qPCR and metaproteomic data – we discuss whether the overcapacity to reduce N2O can be ascribed to gene/protein abundance ratios (nosZ/nir), or whether in-cell competition between the reductases for electrons could be of greater importance.

Impacts of chlorothalonil on denitrification and N2O emission in riparian sediments: Microbial metabolism mechanism

Riparian zones can receive large amounts of nitrate, potentially contributing to water pollution. Denitrification is a major pathway to remove nitrate. Previous research on riparian denitrification focused on natural factors, but frequently neglected the roles of human activity, such as pesticide accumulations. Here, we combined field investigations and exposure experiments to reveal the responses of denitrification and N2O emission to chlorothalonil (CTN, a common pesticide) in column experiments with riparian sediments. In this study, CTN inhibited denitrification and led to nitrate accumulation in sediments. Furthermore, CTN significantly increased N2O emission by 208–377%, and this response was regulated by N2O reductase (NOS) activity rather than nosZ abundance. A mechanistic study indicated that the critical step (glyceraldehyde-3-phosphate to 3-phosphogylcerate) catalyzed by glyceraldehyde-3-phosphate dehydrogenase during microbial metabolism greatly influenced denitrification in CTN-polluted sediments. Our data also revealed that CTN declined electron donor NADH, electron transport system, and denitrifying enzyme activities during denitrification. Such responses suggested that CTN deteriorated sediment denitrification by inhibiting electron production, transport and consumption in denitrifiers. Additionally, structure equation modeling indicated that NOS was the key factor in predicting denitrification rate in CTN-polluted sediments. Overall, this is the first study to explore the effects of pesticide on denitrification and N2O emission in riparian zones at microbial metabolism level. Our results suggest that the safety threshold of CTN accumulation for inhibiting sediment denitrification is approximately 2 mg kg−1, and imply that the wide presence of pesticides in riparian zones could impact eutrophication control of aquatic ecosystems.