Respiration and growth of Paracoccus denitrificans R-1 with nitrous oxide as an electron acceptor

ABSTRACT In the nitrogen biogeochemical cycle, the reduction of nitrous oxide (N2O) to N2 by N2O reductase, which is encoded by nosZ gene, is the only biological pathway for N2O consumption. In this study, we successfully isolated a strain of denitrifying Paracoccus denitrificans R-1 from sewage treatment plant sludge. This strain has strong N2O reduction capability, and the average N2O reduction rate was 5.10 ± 0.11 × 10−9 µmol·h−1·cell−1 under anaerobic condition in a defined medium. This reduction was accompanied by the stoichiometric consumption of acetate over time when N2O served as the sole electron acceptor and the reduction can yield energy to support microbial growth, suggesting that microbial N2O reduction is related to the energy generation process. Genomic analysis showed that the gene cluster encoding N2O reductase of P. denitrificans R-1 was composed of nosR, nosZ, nosD, nosF, nosY, nosL, and nosZ, which was identified as that in other strains in clade I. Respiratory inhibitors test indicated that the pathway of electron transport for N2O reduction was different from that of the traditional electron transport chain for aerobic respiration. Cu2+, silver nanoparticles, O2, and acidic conditions can strongly inhibit the reduction, whereas NO3- or NH4+ can promote it. These findings suggest that modular N2O reduction of P. denitrificans R-1 is linked to the electron transport and energy conservation, and dissimilatory N2O reduction is a form of microbial anaerobic respiration. IMPORTANCE Nitrous oxide (N2O) is a potent greenhouse gas and contributor to ozone layer destruction, and atmospheric N2O has increased steadily over the past century due to human activities. The release of N2O from fixed N is almost entirely controlled by microbial N2O reductase activities. Here, we investigated the ability to obtain energy for the growth of Paracoccus denitrificans R-1 by coupling the oxidation of various electron donors to N2O reduction. The modular N2O reduction process of denitrifying microorganism not only can consume N2O produced by itself but also can consume the external N2O generated from biological or abiotic pathways under suitable condition, which should be critical for controlling the release of N2O from ecosystems into the atmosphere.

N 2 O emissions from soil involve a variety of biological pathways, and it has been estimated that more than 65% of atmospheric N 2 O is derived from microbial N transformations, mainly through the processes of nitrification and denitrification (5).Among them, denitrification is generally considered the largest source of N 2 O, and depending on the types of microorganisms involved and environmental conditions, this process can serve not only as a source of N 2 O but also as a sink for N 2 O (5).Denitrifica tion is the respiratory reduction of nitrogen oxides (NO x ) and enables the survival and reproduction of facultative aerobic bacteria under oxygen-limiting conditions.In this process, nitrate (NO 3 -) is converted into molecular nitrogen (N 2 ) via nitrite (NO 2 -) and the gaseous intermediates, nitric oxide (NO) and nitrous oxide (N 2 O) (6).
In contrast to the large number of N 2 O production pathways and enzymes, only one enzyme is involved in biological N 2 O consumption.This Cu-dependent enzyme is known as N 2 O reductase (nosZ) (7).In typical denitrifying microorganisms (such as Proteobacteria of α-, β-, and γ-classes), NosZ has long been considered the only enzyme that can reduce N 2 O to N 2 , which is called "clade I NosZ." However, an unprecedented nos gene cluster with a novel nosZ containing an additional c-type heme domain at the C terminus was discovered, which was called "clade II NosZ" and which has been identified in a broad range of microbial taxa extending beyond bacteria to archaea (8).According to the current study, clade I NosZ and clade II NosZ are two different phylogenetic groups of the NosZ protein.Additionally, the types of clade II NosZ microorganisms are more complex compared to clade I NosZ microorganisms, and clade II NosZ contains some genes that are not present in clade I NosZ organisms (8,9).
Recently, an increasing number of NosZ-containing microorganisms have been reported to grow via anaerobic N 2 O respiration, with N 2 O as the only electron acceptor, including Bacillus vireti (10), Enifer meliloti 1021 (11), Azospira sp.strain I13 (12), and Gemmatimonas aurantiaca strain T-27 (13).N 2 O respiration is different from the common microbial respiration electron transport chain.The clade I NosZ denitrifying bacteria electron transport chain is located on the membrane by the membrane QCR complex, Q circulation system, cytochrome c, NosZ reductase, and nos gene cluster encoded protein components to form the clade I NosZ electron transport chain system (7).Electron donors can provide the electrons and energy needed for the metabolic activities of microorganisms.In denitrifying bacterial cells, acetate is first converted into acetyl-coA and then directly into the cycletricarboxylic acid (TCA) cycle for utilization, so the utilization rate of acetate is faster than propionate and has a higher denitrification rate (14,15).However, the reduction efficiency of microbial N 2 O by different electron donors remains unclear, and the electron transport chain of N 2 O respiration remains to be explored.There are many factors affecting the environment of microorganisms.Studies have focused on the effects of different factors, including pH, O 2 concentration, N 2 O concentration, or the presence of NO 2 -/NO 3 -on the reduction of N 2 O in strains (16)(17)(18)(19)(20).These factors accelerate or hinder the N 2 O reduction of bacteria mainly through their functional effects on NosZ enzymes or other cell structures.In order to have a more comprehensive understanding of the N 2 O reduction process under different environments, it is necessary to carry out relevant experiments for further research.
In this study, Paracoccus denitrificans R-1, a denitrifying bacterium isolated from the Xinfeng Sewage Plant in Taiwan, was used to explore its N 2 O reduction ability and N 2 O respiratory mechanism.Our results showed that the reduction of N 2 O by P. denitrificans R-1 is a new pathway for N 2 O respiration.

Media, strain, and cultivation
P. denitrificans R-1 was isolated from the sludge of the Taiwan Xifeng Sewage Treatment Plant and stored at the Guangdong Provincial Microbial Strain Preservation Center (GDMCC 1.2910).This strain was stored at −80°C and was pre-cultured aerobically in a nutrient medium (pH 7.0) containing 10 g L −1 NaCl, 5 g L −1 Bacto Peptone, and 5 g L −1 Oxoid Lab-Lemco meat extract at 30°C with shaking at 150 rpm.Then, when the culture reached the exponential phase, it was inoculated into serum bottles of N-free denitrifying medium (N-free DM), which contained 10 g L −1 of Na 2 HPO 4 •12H 2 O, 1.5 g L −1 of KH 2 PO 4 , 0.1 g L −1 of MgSO 4 •7 H 2 O, 4.7 g L −1 of sodium acetate, and 2 mL L −1 of a trace metal solution (21).The cell densities (calibrated with the absorbance value of OD 600 ) of the culture were measured at 600 nm using a spectrophotometer (Shimadzu Enterprise Management Co., LTD).

Incubation
Aerobically grown cells in DM were harvested by centrifugation, washed twice, and resuspended in fresh N-free DM (initial pH 7.5).Then, different organic substances were supplied as electron donors, after which the P. denitrificans R-1 culture was dispensed into 60 mL glass serum bottles (30 mL per bottle).The initial OD 600 of the bacteria in the serum bottles was checked to be about 0.05, and the bottles were crimp-sealed with rubber septa and aluminum caps to ensure an airtight system.The headspace of the serum bottles (30 mL volume) was subsequently replaced with 10% N 2 O (He: N 2 O = 9:1) to analyze the N 2 O reduction capability of P. denitrificans R-1.Organic electron donors (carbon sources) are commonly used by heterotrophic denitrifying bacteria.To explore the difference in the N 2 O reduction ability of different electron donors, a variety of low molecular weight organic compounds were selected as electron donors with a concentration of 10 mM and added to the incubation bottles.Furthermore, the electron donors with better N 2 O reduction effect were selected and added to the incubation bottles at 5 mM concentration to explore the coupling relationship between the electron donor oxidation and the electron acceptor (N 2 O) reduction.The growth of the strain was measured by spectrophotometer.By adding enough rotenone, dicoumarol, and antimycin A, three respiratory inhibitors, we investigated whether complex I or complex II is involved in the electron transport chain during N 2 O reduction.In addition, the effects of temperature, pH, dissolved oxygen, heavy metal ions, and nitrogen substrates (NO 3 and NH 4 + ) on the reduction of N 2 O were conducted.

N 2 O measurement
The N 2 O amount in the incubation bottles was composed of two parts, one in the headspace and the other dissolved in a liquid medium.Three parallel incubations were performed for each sample.After incubation, 50% ZnCl 2 was used to inactivate the bacterial cells.The N 2 O concentration was measured by manually injecting 3-4 mL diluted headspace gas into a gas chromatograph equipped with an HP-PLOT/Q column and an electron capture detector (GC-2014C, Shimadzu Enterprise Management Co., LTD, China).Headspace N 2 O concentration (C G, μmol •L -1 ) in serum bottle is calculated by the following equation 1: (1) Where P is the atmospheric pressure in the serum bottle, C g (ppm; 1 ppm = 1 µmol•mol −1 ) is the concentration of N 2 O in the headspace measured with gas chromatograph (GC), and R is the ideal gas constant, i.e., 0.082057 Latm•(mol•K) −1 .T (K) is the temperature of the water sample at headspace equilibrium.
The N 2 O concentration dissolved in the serum bottle liquid (C L , μmol•L −1 ) is calculated by the following equation 2: (2) denotes the equilibrium constant which can be calculated by Weiss formula (22).
The total amount of N 2 O (Q, μmol) in the serum bottle is calculated by the following equation 3: Where V G and V L are the volumes of gas and liquid, respectively.

Electron donor measurement
Small-molecule organic substances including acetate and lactate were used as electron donors in this study.The cultures were filtered through a 0.22 µm membrane, and the filtrate was used to measure the concentration of electron donors.Acetate and lactate were measured using an ICS-1100 series ion chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a polysulfonate ion-exclusion column (Metrosep A Supp 5).The eluent contained the following: 3.2 mmol L −1 Na 2 CO 3 , 0.8 mM NaHCO 3 , and 3% MeOH.The experiment was performed under the condition of 25°C and 7.3 MPa.

DNA extraction, sequencing, and genomic analysis
P. denitrificans R-1 was inoculated into the nutrient medium and cultured until the logarithmic stage.The bacteria were collected in sterilized centrifuge tubes and stored at −80°C, with a mass of approximately 2 g.Genomic DNA extraction and sequenc ing of this strain were performed by Suzhou GENEWIZ Biotechnology Co., Ltd.After obtaining high-quality genomic DNA, the fragments were randomly broken down into the corresponding length fragments to construct a library.The qualified libraries were sequenced (150 bp paired-end sequencing) on the NovaSeq system.Detailed informa tion regarding genomic sequencing can be found in our previous study (23).
The genome analysis process consisted of four steps: (i) data quality control: preprocessing of the original data obtained by sequencing.Low-quality data were filtered, and splice sequences were removed to prevent low-quality data from having a negative impact on subsequent analyses.Clean data obtained after data preprocess ing were used for subsequent analyses.The software used for the quality statistics of the second-generation sequencing data was adapted (v 1.9.1).(ii) Genome assembly: HGAP4 software was used to assemble the third-generation sequencing data.After the assembly was completed, the quality control of the second-generation sequencing data was compared with that of the third-generation assembly results, and the final assembly results were obtained using Pilon.(iii) Prediction of coding and non-coding genes: after assembly, coding and non-coding RNAs were predicted using the Prodigal software (v3.02).(iv) Gene function annotation: the predicted protein sequence of the coding gene was compared with the protein sequences contained in each database.The databases used in this study mainly included Cluster of Orthologous Groups, Gene Ontology, Carbohydrate-Active Enzymes Database, and Kyoto Encyclopedia of Genes and Genomes.

Statistical analyses
The statistical analyses in this study were performed with the Origin 2018 software.The strain information was collected on NCBI, the phylogenetic tree was constructed by selecting Neighbor-Joining method with Mega 7.0 software, and the gene clusters were drawn with ChiPlot.Unless otherwise stated, the experiment was performed in triplicate, and the mean of triplicate samples was taken to represent data points and the SD of triplicate samples to represent error values.

RESULTS N 2 O reduction coupled to oxidation of electron donors
To explore the relationship between N 2 O reduction and electron donor oxidation, changes in the concentrations of both electron donors and N 2 O in the culture system were analyzed simultaneously.Sodium acetate and sodium lactate with better N 2 O reduction efficiency were selected as electron donors, and the N 2 O reduction efficiency of different electron donors on P. denitrificans R-1 is shown in Fig. S1.In the culture with sodium acetate as the electron donor, the amount of N 2 O decreased from the initial 83.81 ± 1.65 µmol to 0.58 ± 0.24 µmol after 16 h of culturing, and N 2 O decreased by 83.23 ± 1.89 µmol.In addition, sodium acetate decreased from 173.45 ± 4.77 µmol to 78.84 ± 12.44 µmol during culture, and sodium acetate decreased by 94.61 ± 17.21 µmol (Fig. 1A).In the culture with sodium lactate as the electron donor, the amount of N 2 O decreased from the initial 89.63 ± 0.28 µmol to 0.77 ± 0.03 µmol at 16 h, N 2 O decreased by 88.86 ± 0.31 µmol, sodium lactate decreased from 105.99 ± 8.28 µmol to 63.13 ± 0.74 µmol in 0-16 h, and sodium lactate decreased by 42.86 ± 7.54 µmol (Fig. 1B).According to the fitting equations (R 2 = 0.9764 for acetate and R 2 = 0.9485 for lactate), there was a significant linear relationship between the reduction of N 2 O and the oxidation of the electron donors of acetate or lactate.

Growth of P. denitrificans R-1 with N 2 O as sole electron acceptor
In a culture experiment using sodium acetate as the electron donor and N 2 O as the sole electron acceptor, P. denitrificans R-1 exhibited N 2 O consumption and growth (Fig. 2).The consumption of N 2 O was slow from 0 h to 4 h, and N 2 O decreased rapidly from 4 h to 20 h.The N 2 O virtually remained unchanged from 20 h to 24 h.In this process, the N 2 O decreased from the initial 83.78 ± 2.88 µmol to 5.47 ± 0.72 µmol, and the average N 2 O consumption rate was 5.10 ± 0.11 × 10 −9 µmol•h −1 •cell −1 .However, no significant change in the amount of N 2 O was observed in the no-cell incubation, indicating that the reduction of N 2 O in the experimental group was not caused by spontaneous degradation or transformation of N 2 O but was consumed by P. denitrificans R-1.No N 2 O release was observed during culturing.Furthermore, N 2 O reduction was accompanied by the growth of P. dienitrificans R-1 in the culture.The growth was slow during 0-4 h but became relatively faster after 4 h and remained stable for 20-24 h (Fig. 2).The OD 600 value of P. denitrificans R-1 increased from 0.0675 ± 0.0021 to 0.1925 ± 0.0007 within 24 h; with an increase of 0.125 ± 0028, exponential growth rate is 0.0437 ± 0.0015 h −1 (Table S1).The OD 600 value virtually did not change within 24 h in the control group without N 2 O, suggesting that the growth of P. denitrificans R-1 was due to energy conservation during N 2 O reduction.

Effect of respiratory inhibitors on N 2 O reduction
To explore the electron transport chain components that are possibly involved in N 2 O reduction by P. denitrificans R-1, three respiratory inhibitors (dicoumarol, rotenone, and antimycin A) were used to explore the effect of N 2 O reduction by P. denitrificans R-1.Among these, dicoumarol inhibits electron transport from vitamin K to quinones, rotenone blocks electron transport from NADH to CoQ, and antimycin A inhibits electron transport from QH 2 to cytochrome C 1 (24).The concentration range of dicoumarol and rotenone inhibitors was pre-tested (Fig. S2), and the concentration of antimycin A was sufficient.All three inhibitors had no significant effect on the N 2 O reduction process by P. denitrificans R-1 (Fig. 3), suggesting that N 2 O reduction by P. denitrificans R-1 had different electron transport components from the traditional one involving complexes I and II.To determine which electron transport components were involved in N 2 O reduction by this strain, further study using new methods is necessary.

Composition and characteristics of gene cluster of P. dienitrificans R-1
A phylogenetic evolutionary tree was constructed using nosZ sequences from different bacterial strains collected from the NCBI database (Fig. 4).The evolutionary tree was divided into two clusters, clade I and clade II, with P. denitrificans R-1 (bold) distributed in clade I.The differences between clades I and II are not only reflected in the phylogeny of the NosZ protein but also in the composition and structure of their respective nos gene clusters (25).The genomic locus encoding NosZ is a part of the nos gene cluster, which also includes genes encoding helper proteins required for the maturation and function of NosZ (8).P. denitrificans R-1 nos gene cluster consisted of nosR, nosZ, nosD, nosF, nosY, and nosL, which is a common pattern in nos gene cluster of clade I microor ganisms.However, clade II microorganisms have more complex and diverse nos gene clusters than those of clade I microorganisms (Fig. 5).Because nosD, -F, and -Y are the strongest conserved genes in nos gene clusters, they exist in both clade I and clade II type microorganisms (26).
N 2 O oxidoreductase (NosZ) was thought to be sensitive to O 2 (27).Three gradient concentrations of O 2 (5%, 10%, and 20%) were selected for culturing P. denitrificans R-1.No N 2 O consumption was observed after 24 h culture under all three gradient concentra tions of O 2 (Fig. 5E), suggesting that the O 2 can strongly inhibit the N 2 O reduction of P. denitrificans R-1.The growth of the strain was also inhibited in the presence of oxygen (Fig. 5F; Table S4).

Effects of NO 3 − and NH 4 + on N 2 O reduction
In the culture where different concentrations of NO 3 -were added, the N 2 O reduction rate by P. denitrificans R-1 increased (Fig. 6A).The average N 2 O reduction rate by P. denitrificans R-1 was 1.53 ± 0.69 × 10 −8 µmol•h −1 •cell −1 , which was approximately 3.5 times higher than that in the culture without NO 3 -(4.39± 0.13 × 10 −9 µmol•h −1 •cell −1 ).This suggests that the N 2 O reduction of P. denitrificans R-1 can be promoted by NO 3 -in the culture.Moreover, in the culture with NO 3 -(except for the addition of 20 mg•L −1 NO 3 -), the exponential growth rate of P. denitrificans R-1 was higher than that in the culture without NO 3 -(Fig.6B; Table S5), possibly because the strain can use NO 3 -as an electron acceptor to obtain energy for growth.Similarly, the N 2 O reduction capability of P. denitrificans R-1 improved in cultures with different concentrations of NH 4 + (Fig. 6C).The average N 2 O reduction rate by P. denitrificans R-1 was 1.39 ± 0.86 × 10 −8 µmol•h −1 •cell −1 , which was approximately three times higher than that in the culture without NH 4 + (4.39 ± 0.13 × 10 −9 µmol•h −1 •cell −1 ).The exponential growth rate of strain with NH 4 + added was significantly higher than that without NH 4 + (Fig. 6D; Table S5).Therefore, both NO 3 -and NH 4 + in the culture improved N 2 O reduction by P. denitrificans R-1.

Effects of heavy metal ions on N 2 O reduction
Trace amounts of Cu are believed to promote microbial growth, and this element is an important component of nosZ gene (7).With the development and application of nanomaterials, silver nanoparticles (AgNPs) have become the most widely used owing to their superior bactericidal abilities (28).Two types of heavy-metal ions, Cu 2+ and AgNPs, were selected to analyze their effects on N 2 O reduction.When the concentration of Cu 2+ was 10 mg•L −1 , the N 2 O reduction rate decreased to 2.46 ± 0.35 × 10 −9 µmol•h −1 •cell −1 compared to the rate of 5.42 ± 0.01 × 10 −9 µmol•h −1 •cell −1 in the culture without Cu 2+ .When the concentration of Cu 2+ increased to 50 mg•L −1 and 100 mg•L −1 , the reduction of N 2 O was not completely detected (Fig. 6E).These results demonstrate that Cu 2+ strongly donor oxidation and electron acceptor reduction in N 2 O reduction process.Linear fitting analysis also confirmed that the oxidation of the electron donor and the reduction of the electron acceptor showed a typical coupling relationship (R 2 > 0.9; Fig. 1).When sodium acetate and sodium lactate are used as electron donors, the reduction process of N 2 O conforms to the chemical equation in Table 1.Theoretically, 1 mol of sodium acetate can provide 8 mol of electrons and support 4 mol of N 2 O reduction.One mol of sodium lactate can provide 12 mol of electrons, supporting 6 mol N 2 O reduction.The ratios of the acetic acid and sodium lactate by P. denitrificans R-1 to the amount of N 2 O reduced were 1:0.89 and 1:2.07.The bioavailability efficiency of sodium acetate and sodium lactate reached 22.25% and 34.50%, respectively.This result indicates that the oxidation of acetate or lactate was sufficient for energy conservation when N 2 O was completely reduced to N 2 .The redox potentials of various electron donors are related to the different N 2 O reduction efficiencies.Generally, the electron transfer in organisms is from the direction of low redox potential to the direction of high, and the higher the difference between potentials, the higher the conversion efficiency.Of course, it is also related to the specificity of the reductase in the organism.In the process of N 2 O reduction, the potential difference between sodium lactate redox is higher than that of sodium acetate.Our study also confirmed that sodium lactate as an electron donor has higher conversion efficiency than sodium acetate.

Energy conservation from dissimilatory N 2 O reduction by P. denitrificans R-1
Cell growth depends on the supply of energy and nutrients.In contrast to previous reports (7), the growth of P. denitrificans R-1 was observed only when N 2 O was reduced as the sole electron acceptor.The OD 600 value increased from 0.0675 to 0.1925 within 24 h of incubation (Table S1), suggesting that the coupled oxidation of acetate to N 2 O reduction provided energy for the growth and metabolism of P. denitrificans R-1.A previous study showed that some denitrifiers can grow by N 2 O reduction using N 2 O as the sole electron acceptor, known as N 2 O respiration bacteria (NRBs).Most of these bacteria are facultative anaerobes and harbor a clade II N 2 O reductase, including Wolinella succinogenes (29), Campylobacter fetus (30), Anaeromyxobacter dehalogenans (8), B. vireti (10), Dechloromonas aromatica (31), Dechloromonas denitrificans (31), and Azospira sp.strain I13 (12).However, N 2 O respiration is widely underexplored, and only a few NRBs of traditional denitrifying bacteria with clade I N 2 O reductase have been shown to grow through N 2 O reduction.Some NRBs possess nrfA, a key functional gene for dissimilatory nitrate reduction to ammonium, suggesting that N 2 O reduction is coupled with nitrogen fixation, in which N 2 O is first reduced to N 2 , and then N 2 is further reduced to ammonium nitrogen and integrated into the cell biomass (32).In addition, Park et al. showed that Guarianthe auruantiaca T-27 was able to reduce N 2 O when O 2 was depleted, and O 2 was initially present, but no growth was observed (13).A plausible explanation for this lack of growth is that obligate aerobic microorganisms with nosZ may utilize N 2 O as a temporary surrogate for O 2 to survive periodic anoxia.In the present study, our results suggest that P. denitrificans R-1, a traditional denitrifier with clade I N 2 O reductase, can grow when N 2 O is reduced coupled with electron oxidation; therefore, P. denitrificans R-1 can be called an NRB.

Electron transportation system for microbial N 2 O reduction
Respiratory inhibitor experiments showed that the electron transfer of P. denitrificans R-1 in the N 2 O reduction process did not involve the conventional respiratory electron transfer enzyme complexes I and II (Fig. 3).Previous studies have reported that the electron transport chain of classic nosZ-I type of denitrifying bacteria is located on the cell membrane, including QCR compounds, the Q circulation system, cytochrome C, nosZ reductase, and nos genes encoding proteins (NosR, -X -C, -D, F, -Y, and -L) (7).Genomic data showed that P. denitrificans R-1 has a similar nos gene cluster (NosR, -Z, -D, -F, -Y, and -L; Fig. 4), suggesting that P. denitrificans R-1 has electron-transfer protein components similar to those of classic NosZ-I-type denitrifying bacteria.
It has been shown that three proteins, NosD, NosY, and NosF, encoded by nos gene clusters, may constitute a complex transporter that binds to the cell membrane and couples ATP hydrolysis; however, it is unclear whether they can play the role of transport ers (7,26,33).NosL is a Cu-containing outer membrane lipoprotein that is closely related to the NosDYF complex.Studies have suggested that NosL may provide Cu for NosZ (26).NosR also participates in electron transfer.FeS and flavin mononucleotide (FMN) are distributed at both ends of this protein and can transfer low-potential electrons from the cytoplasm across membranes to NosZ located in the pericytoplasm, which is an electron transfer pathway independent of Qcr (7).NosX is a signal peptide containing a Tat sequence that exists in the periplasmic space and contains a flavin protein with flavin adenine dinucleotide (FAD)as a co-group.NosX is mainly involved in the biogenesis of NosR and is a cofactor of the FMN terminal of NosR.NosX is associated with the influence of ApbE proteins in Fe-S centers, and ApbE has been shown to be a flavin donor in NosR (7,34).
Based on inhibitor experiments and genomic analysis, we deduced an electron transfer model for the N 2 O reduction growth of P. denitrificans R-1 (Fig. 7).Electrons produced by the conversion of acetic acid are transferred through the electron transport chain, generating an electrochemical force on the membrane and driving ATP synthesis.However, the exact mechanisms underlying microbial N 2 O respiration remain unclear.

Potential ecological significance of microbial N 2 O reduction
Denitrification pathways are highly modular.Reduction of N 2 O by typical denitrifying bacteria occurs after the production of N 2 O.Moreover, the reduction of N 2 O is an independent process (33).Our study showed that the nosZ type I bacterium, P. denitrifi cans R-1, can respire N 2 O as the sole electron donor.
In addition, the single-factor regulation experiment was used to further reveal the effect of different factors on the N 2 O reduction process of P. denitrificans R-1.Low temperature often leads to a decrease in enzyme activity, which affects cell growth and metabolism.At the same time, low temperature also leads to delayed expression of denitrifying genes (35).The N 2 O reduction capacity of P. denitrificans R-1 also increased with the increase in temperature.The N 2 O reduction capacity of P. denitrificans R-1 was enhanced under alkaline conditions and inhibited under acidic conditions.This result is similar to the result of the study by Saleh-Lakha et al. (35), which shows that when pH = 5, the expression of denitrification gene in Pseudomonas mandelii is the most unfavorable.The presence of O 2 is not conducive to the N 2 O reduction of P. denitrificans R-1.This is consistent with most current research results (36)(37)(38), nosZ is an oxygen-sensitive gene, denitrifying bacteria cannot continue to catalyze the last step of denitrification process (N 2 O reduction to N 2 ) under aerobic conditions, so the final product is N 2 O rather than N 2 .After NO 3 -/NH 4 + was added to P. denitrificans R-1 as an additional electron acceptor, the N 2 O reduction capacity of P. denitrificans R-1 was greatly improved.The study of Marques et al. (39) showed that the metabolism of NO 3 produced higher ATP than N 2 O reduction process, so it could provide sufficient energy for the process of cell reduction of external N 2 O.The addition of NO 3 -can promote the denitrification and the electron transport and the activities of related enzymes.Because the pathway of N 2 O reduction is a part of denitrification, the N 2 O consumption would be accelerated by adding the NO 3 -in the medium.Ammonium assimilation is more beneficial for the growth and propagation of bacteria and then convenient for the N 2 O reduction.The presence of Cu 2+ can inhibit the N 2 O reduction of P. denitrificans R-1.It has been reported (28) that appropriately increasing Cu 2+ concentration (0-0.05mg•L −1 ) can promote the expression of nosZ gene in denitrifiers Pseudomonas stutzeri PCN-1.The high concentration of Cu 2+ (0.5-5 mg•L −1 ) would inhibit the denitrification activity and gene expression of the strain, resulting in more N 2 O emission.In this experiment, it was found that the N 2 O reduction efficiency of P. denitrificans R-1 gradually decreased at the concentration of 10-100 mg•L −1 of Cu 2+ , and whether it would promote the reduction of N 2 O at a lower concentration of Cu 2+ needs further investigation.AgNPs reduced the denitrification efficiency by inhibiting the expression of denitrification genes and breaking the cell membrane (40,41).Interestingly, we found that the reduction efficiency of N 2 O was enhanced at low concentrations of AgNPs.It is hypothesized that AgNPs oxidize in water to consume oxygen and enhance the expression of the oxygen-sensitive nosZ gene.
In summary, the modular N 2 O reduction process of typical denitrifying bacteria (nosZ-I) not only can consume N 2 O produced by themselves but can also consume the external N 2 O generated from nondenitrification biological or abiotic pathways under suitable conditions.The exploration of N 2 O respiration of P. denitrificans R-1 contributes to further understanding of the regulatory role of microorganisms on N 2 O in the natural environment.This is essential for controlling N 2 O emissions using microorganisms.on the N 2 O reduction of P. denitrificans R-1.Table S1: Growth of Paracoccus denitrificans R-1 in culture with N2O as the only electron acceptor.Table S2: Growth of Paracoccus denitrificans R-1 at different temperatures.Table S3: Growth of Paracoccus denitrificans R-1 at different pH.Table S4: Growth of Paracoccus denitrificans R-1 at different O 2 concentration.Table S5: Growth of Paracoccus denitrificans R-1 with additional nitrogen sources.Table S6: N 2 O consumption rate of Paracoccus denitrificans R-1 in 8 h cultured with different electron donors.

FIG 2
FIG 2 Growth of P. denitrificans R-1 during N 2 O reduction.In control vessels without N 2 O, cell numbers did not increase.No N 2 O reduction occurred in control cultures that received no inoculum.•, N 2 O; ■, He, no cells; ▲, cells = OD 600 nm; ○, N 2 O, no cells; △, cells = OD 600 nm, no N 2 O. Data points are averages of duplicate experiments, and error bars represent SDs.

FIG 3
FIG 3 Respiratory inhibition experiment of P. denitrificans R-1.Data points are averages of duplicate experiments, and error bars represent SDs.

FIG 5 FIG 6
FIG 5 Effects of temperature, pH, and O 2 on N 2 O reduction and growth of P. denitrificans R-1.N 2 O consumption curve (A) and strain growth curve (B) at different temperatures.N 2 O consumption curve (C) and strain growth curve (D) at different pH values.N 2 O consumption curve (E) and strain growth curve (F) under different O 2 concentrations.Data points are averages of duplicate experiments, and error bars represent SDs.