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.

INTRODUCTION

Nitrous oxide (N₂O), a colorless, stable gas, has an average lifetime of 120 years in the atmosphere and is ultimately decomposed by ultraviolet light (1). As one of the most important forms of nitrogen pollution, N₂O is currently the third largest greenhouse gas (GHG) emitted and the largest anthropogenic stratospheric ozone-depleting substance (2). Although N₂O only accounts for approximately 0.03% of total GHG emissions, it has a nearly 300-fold greater potential for global warming based on its radiative capacity compared to that of carbon dioxide (CO₂) (3, 4). Therefore, controlling N₂O emissions is essential for curbing global warming and climate change.

N₂O emissions from soil involve a variety of biological pathways, and it has been estimated that more than 65% of atmospheric N₂O 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 N₂O, and depending on the types of microorganisms involved and environmental conditions, this process can serve not only as a source of N₂O but also as a sink for N₂O (5). Denitrification 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 (NO₃^-) is converted into molecular nitrogen (N₂) via nitrite (NO₂^-) and the gaseous intermediates, nitric oxide (NO) and nitrous oxide (N₂O) (6).

In contrast to the large number of N₂O production pathways and enzymes, only one enzyme is involved in biological N₂O consumption. This Cu-dependent enzyme is known as N₂O 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 N₂O to N₂, 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 N₂O respiration, with N₂O 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). N₂O 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 N₂O by different electron donors remains unclear, and the electron transport chain of N₂O 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, O₂ concentration, N₂O concentration, or the presence of NO₂^- /NO₃^- on the reduction of N₂O in strains (16–20). These factors accelerate or hinder the N₂O 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 N₂O 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 N₂O reduction ability and N₂O respiratory mechanism. Our results showed that the reduction of N₂O by P. denitrificans R-1 is a new pathway for N₂O respiration.

MATERIALS AND METHODS

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 Na₂HPO₄·12H₂O, 1.5 g L⁻¹ of KH₂PO₄, 0.1 g L⁻¹ of MgSO₄·7 H₂O, 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 OD₆₀₀) 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 OD₆₀₀ 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% N₂O (He: N₂O = 9:1) to analyze the N₂O 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 N₂O 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 N₂O 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 (N₂O) 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 N₂O reduction. In addition, the effects of temperature, pH, dissolved oxygen, heavy metal ions, and nitrogen substrates (NO₃^- and NH₄⁺) on the reduction of N₂O were conducted.

N₂O measurement

The N₂O 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% ZnCl₂ was used to inactivate the bacterial cells. The N₂O 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 N₂O concentration (C_G, μmol ·L^-1) in serum bottle is calculated by the following equation 1:

$${\displaystyle {\mathrm{C}}_{\mathrm{G}}={\displaystyle \frac{\mathrm{P}\times {\mathrm{C}}_{\mathrm{g}}}{1013.25\times \mathrm{R}\times \mathrm{T}}}}$$ (1)

Where P is the atmospheric pressure in the serum bottle, C_g (ppm; 1 ppm = 1 µmol·mol⁻¹) is the concentration of N₂O 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 N₂O concentration dissolved in the serum bottle liquid (C_L, μmol·L⁻¹) is calculated by the following equation 2:

$$C_L=C_G\times (K_0\times R\times T)$$ (2)

K₀ [mol·(L·atm) ⁻¹] denotes the equilibrium constant which can be calculated by Weiss formula (22).

The total amount of N₂O (Q, μmol) in the serum bottle is calculated by the following equation 3:

$$Q=C_G\times V_G+C_L\times V_L$$ (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 Na₂CO₃, 0.8 mM NaHCO₃, 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 sequencing 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 information 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 preprocessing 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

N₂O reduction coupled to oxidation of electron donors

To explore the relationship between N₂O reduction and electron donor oxidation, changes in the concentrations of both electron donors and N₂O in the culture system were analyzed simultaneously. Sodium acetate and sodium lactate with better N₂O reduction efficiency were selected as electron donors, and the N₂O 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 N₂O decreased from the initial 83.81 ± 1.65 µmol to 0.58 ± 0.24 µmol after 16 h of culturing, and N₂O 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 N₂O decreased from the initial 89.63 ± 0.28 µmol to 0.77 ± 0.03 µmol at 16 h, N₂O 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 (R² = 0.9764 for acetate and R² = 0.9485 for lactate), there was a significant linear relationship between the reduction of N₂O and the oxidation of the electron donors of acetate or lactate.

Fig 1.

P. denitrificans R-1 for N₂O reduction coupled with electron donor oxidation. Sodium acetate as electron donor (A) and sodium lactate as electron donor (B). Data points are averages of duplicate experiments, and error bars represent SDs.

Growth of P. denitrificans R-1 with N₂O as sole electron acceptor

In a culture experiment using sodium acetate as the electron donor and N₂O as the sole electron acceptor, P. denitrificans R-1 exhibited N₂O consumption and growth (Fig. 2). The consumption of N₂O was slow from 0 h to 4 h, and N₂O decreased rapidly from 4 h to 20 h. The N₂O virtually remained unchanged from 20 h to 24 h. In this process, the N₂O decreased from the initial 83.78 ± 2.88 µmol to 5.47 ± 0.72 µmol, and the average N₂O consumption rate was 5.10 ± 0.11 × 10⁻⁹ µmol·h⁻¹·cell⁻¹. However, no significant change in the amount of N₂O was observed in the no-cell incubation, indicating that the reduction of N₂O in the experimental group was not caused by spontaneous degradation or transformation of N₂O but was consumed by P. denitrificans R-1. No N₂O release was observed during culturing. Furthermore, N₂O 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 OD₆₀₀ 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 OD₆₀₀ value virtually did not change within 24 h in the control group without N₂O, suggesting that the growth of P. denitrificans R-1 was due to energy conservation during N₂O reduction.

Fig 2.

Growth of P. denitrificans R-1 during N₂O reduction. In control vessels without N₂O, cell numbers did not increase. No N₂O reduction occurred in control cultures that received no inoculum. ●, N₂O; ■, He, no cells; ▲, cells = OD₆₀₀ nm; ○, N₂O, no cells; △, cells = OD₆₀₀ nm, no N₂O. Data points are averages of duplicate experiments, and error bars represent SDs.

Effect of respiratory inhibitors on N₂O reduction

To explore the electron transport chain components that are possibly involved in N₂O reduction by P. denitrificans R-1, three respiratory inhibitors (dicoumarol, rotenone, and antimycin A) were used to explore the effect of N₂O 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 QH₂ to cytochrome C₁ (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 N₂O reduction process by P. denitrificans R-1 (Fig. 3), suggesting that N₂O 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 N₂O reduction by this strain, further study using new methods is necessary.

Fig 3.

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

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 microorganisms. 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).

Fig 4.

Phylogenetic tree based on NosZ protein sequence and comparison of nos gene clusters in different clades of NosZ. For each gene cluster, nosZ and its accessory genes (BDFGHLRXY) are labeled and colored according to homology across different gene clusters. Additional proteins, including iron-sulfur-binding proteins (FeS), Rieske iron-sulfur proteins (S), and b- and c-type cytochromes (Cyb and Cyc, respectively) are also labeled. Non-colored genes denote open reading frames with no orthologs in other nos gene clusters, and the scale bar at the lower-right indicates gene size.

Fig 5.

Effects of temperature, pH, and O₂ on N₂O reduction and growth of P. denitrificans R-1. N₂O consumption curve (A) and strain growth curve (B) at different temperatures. N₂O consumption curve (C) and strain growth curve (D) at different pH values. N₂O consumption curve (E) and strain growth curve (F) under different O₂ concentrations. Data points are averages of duplicate experiments, and error bars represent SDs.

Effects of temperatures, pH, and O₂ on N₂O reduction

The average N₂O reduction rate of P. denitrificans R-1 was 2.00 ± 0.30 × 10⁻⁹ µmol·h⁻¹·cell⁻¹ (0–24 h), 5.50 ± 0.53 × 10⁻⁹ µmol·h⁻¹·cell⁻¹ (0–24 h), and 1.17 ± 0.30 × 10⁻⁸ µmol·h⁻¹·cell⁻¹ (0–18 h) under the condition of 20°C, 30°C, and 40°C, respectively (Fig. 5A). The OD₆₀₀ value for growth of P. denitrificans R-1 was 0.0365 ± 0.0063, 0.1215 ± 0.0064, and 0.0175 ± 0.0233 at 20°C, 30°C, and 40°C, respectively; the exponential growth rate was the highest at 30°C (Fig. 5B; Table S2). These results suggest that the N₂O reduction rate of P. denitrificans R-1 was promoted with an increase of temperature in a limited range, but the growth of strain was most obvious at 30°C.

At pH 5.0 and 6.0, no N₂O consumption or cell growth was observed within 24 h, suggesting that the reduction of N₂O by P. denitrificans R-1 was not favorable under acidic conditions (Fig. 5C). At pH 7.0, 8.0, and 9.0, P. denitrificans R-1 was able to reduce N₂O normally (Fig. 5C), indicating that the N₂O-reducing ability of the strain was activated under neutral or alkaline conditions. Growth was optimal at pH 7.0, but it was inhibited under acidic or alkaline conditions (Fig. 5D; Table S3).

N₂O oxidoreductase (NosZ) was thought to be sensitive to O₂ (27). Three gradient concentrations of O₂ (5%, 10%, and 20%) were selected for culturing P. denitrificans R-1. No N₂O consumption was observed after 24 h culture under all three gradient concentrations of O₂ (Fig. 5E), suggesting that the O₂ can strongly inhibit the N₂O 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 NO₃⁻ and NH₄⁺ on N₂O reduction

In the culture where different concentrations of NO₃^- were added, the N₂O reduction rate by P. denitrificans R-1 increased (Fig. 6A). The average N₂O 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 NO₃^- (4.39 ± 0.13 × 10⁻⁹ µmol·h⁻¹·cell⁻¹). This suggests that the N₂O reduction of P. denitrificans R-1 can be promoted by NO₃^- in the culture. Moreover, in the culture with NO₃^- (except for the addition of 20 mg·L⁻¹ NO₃^-), the exponential growth rate of P. denitrificans R-1 was higher than that in the culture without NO₃^- (Fig. 6B; Table S5), possibly because the strain can use NO₃^- as an electron acceptor to obtain energy for growth. Similarly, the N₂O reduction capability of P. denitrificans R-1 improved in cultures with different concentrations of NH₄⁺ (Fig. 6C). The average N₂O 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 NH₄⁺ (4.39 ± 0.13 × 10⁻⁹ µmol·h⁻¹·cell⁻¹). The exponential growth rate of strain with NH₄⁺ added was significantly higher than that without NH₄⁺ (Fig. 6D; Table S5). Therefore, both NO₃^- and NH₄⁺ in the culture improved N₂O reduction by P. denitrificans R-1.

Fig 6.

Effects of adding different nitrogen substrates and heavy metal ions on N₂O reduction and growth of P. Denitrificans R-1. After the addition of metal ions, turbidity and color of the medium had an impact on OD₆₀₀ determination, so OD₆₀₀ value of growth with the addition of heavy metal ions was not measured. N₂O-reduction process curve (A) and growth curve (B) of P. denitrificans R-1 under different concentrations of NO₃^-. N₂O-reduction process curve (C) and growth curve (D) of P. denitrificans R-1 under different concentrations of NH₄⁺. N₂O-reduction at different Cu²⁺ concentrations (E). N₂O-reduction at different silver nanoparticles (AgNPs) concentrations (F). Data points are averages of duplicate experiments with error bars representing SDs.

Effects of heavy metal ions on N₂O 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, Cu²⁺ and AgNPs, were selected to analyze their effects on N₂O reduction. When the concentration of Cu²⁺ was 10 mg·L⁻¹, the N₂O 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 Cu²⁺. When the concentration of Cu²⁺ increased to 50 mg·L⁻¹ and 100 mg·L⁻¹, the reduction of N₂O was not completely detected (Fig. 6E). These results demonstrate that Cu²⁺ strongly inhibited N₂O reduction of P. denitrificans R-1. In contrast, the reduction rate of N₂O increased from 5.42 ± 0.01 × 10⁻⁹ µmol·h⁻¹·cell⁻¹ to 1.04 ± 0.35 × 10⁻⁸ µmol·h⁻¹·cell⁻¹ when the concentration of AgNPs was 5 mg·L⁻¹ (Fig. 6F), indicating that a low concentration of AgNPs was able to promote N₂O reduction by P. denitrificans R-1. However, N₂O reduction was greatly reduced when the concentration of AgNPs was increased to 10 or 20 mg·L⁻¹ (Fig. 7F), suggesting that the high concentration of AgNPs inhibited N₂O reduction by P. denitrificans R-1.

Fig 7.

P. denitrificans R-1 N₂O respiratory electron transport model using sodium acetate as an electron donor. For simplicity, only major enzymes are shown. Electron transfer chain also includes nosD, Y, L, X, and R which are not shown, and unknown enzymes cannot be ruled out.

DISCUSSION

Oxidation of electron donor coupled to N₂O reduction by P. denitrificans R-1

Microbial N₂O respiration is an important process in the N cycle. Many microorganisms respond to N₂O as an electron acceptor (10, 11, 13, 16). In the current study, we found that P. denitrificans R-1 could reduce N₂O via the oxidation of electron donors. Sodium acetate, ethanol, sodium propionate, sodium pyruvate, sodium lactate, sodium succinate, and glucose acted as effective electron donors to support N₂O reduction by P. denitrificans R-1 (Fig. S1). Under the first 8 h of culture, sodium acetate, sodium lactate, and ethanol have higher N₂O reduction rate, among which sodium acetate has the highest N₂O reduction rate and is the strongest electron donor (Table S6). According to previous studies (14, 15), acetate can be converted into acetyl-CoA and then directly integrated into the TCA cycle for degradation in denitrifying bacterial cells; therefore, the utilization rate of acetate is more efficient than that of other electron donors, resulting in a higher N₂O reduction rate. We select sodium acetate and sodium lactate with high N₂O reduction rate to further explore the coupling relationship between electron donor oxidation and electron acceptor reduction in N₂O 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 (R² > 0.9; Fig. 1).

When sodium acetate and sodium lactate are used as electron donors, the reduction process of N₂O 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 N₂O reduction. One mol of sodium lactate can provide 12 mol of electrons, supporting 6 mol N₂O reduction. The ratios of the acetic acid and sodium lactate consumed by P. denitrificans R-1 to the amount of N₂O 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 N₂O was completely reduced to N₂. The redox potentials of various electron donors are related to the different N₂O 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 N₂O 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.

TABLE 1.

Theoretical and actual ratios of electron donor to acceptor in the oxidation-reduction reaction

Electron donor	Electron acceptor	Oxidation-reduction reaction	Ratio of donor to acceptor (mole/mole)
			Theoretical	Experimental	Efficiency
Acetate	Nitrous oxide	$\frac{1}{8}{CH}_{3}CO{O}^{-}+\frac{1}{2}{N}_{2}O\to \frac{1}{8}C{O}_2+\frac{1}{8}HC{O}_3+\frac{1}{2}{N}_2+\frac{3}{8}{H}_{2}O$	1:4	1:0.89	22.25%
Lactate		$\frac{1}{12}{CH}_{3}CHOHCO{O}^{-}+\frac{1}{2}{N}_{2}O\to \frac{1}{6}C{O}_2+\frac{1}{12}HC{O}_3+\frac{1}{2}{N}_2+\frac{1}{6}{H}_{2}O$	1:6	1:2.07	34.50%

Energy conservation from dissimilatory N₂O 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 N₂O was reduced as the sole electron acceptor. The OD₆₀₀ value increased from 0.0675 to 0.1925 within 24 h of incubation (Table S1), suggesting that the coupled oxidation of acetate to N₂O reduction provided energy for the growth and metabolism of P. denitrificans R-1. A previous study showed that some denitrifiers can grow by N₂O reduction using N₂O as the sole electron acceptor, known as N₂O respiration bacteria (NRBs). Most of these bacteria are facultative anaerobes and harbor a clade II N₂O 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, N₂O respiration is widely underexplored, and only a few NRBs of traditional denitrifying bacteria with clade I N₂O reductase have been shown to grow through N₂O reduction. Some NRBs possess nrfA, a key functional gene for dissimilatory nitrate reduction to ammonium, suggesting that N₂O reduction is coupled with nitrogen fixation, in which N₂O is first reduced to N₂, and then N₂ 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 N₂O when O₂ was depleted, and O₂ 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 N₂O as a temporary surrogate for O₂ to survive periodic anoxia. In the present study, our results suggest that P. denitrificans R-1, a traditional denitrifier with clade I N₂O reductase, can grow when N₂O is reduced coupled with electron oxidation; therefore, P. denitrificans R-1 can be called an NRB.

Electron transportation system for microbial N₂O reduction

Respiratory inhibitor experiments showed that the electron transfer of P. denitrificans R-1 in the N₂O 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 transporters (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 N₂O 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 N₂O respiration remain unclear.

Potential ecological significance of microbial N₂O reduction

Denitrification pathways are highly modular. Reduction of N₂O by typical denitrifying bacteria occurs after the production of N₂O. Moreover, the reduction of N₂O is an independent process (33). Our study showed that the nosZ type I bacterium, P. denitrificans R-1, can respire N₂O as the sole electron donor.

In addition, the single-factor regulation experiment was used to further reveal the effect of different factors on the N₂O 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 N₂O reduction capacity of P. denitrificans R-1 also increased with the increase in temperature. The N₂O 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 O₂ is not conducive to the N₂O reduction of P. denitrificans R-1. This is consistent with most current research results (36–38), nosZ is an oxygen-sensitive gene, denitrifying bacteria cannot continue to catalyze the last step of denitrification process (N₂O reduction to N₂) under aerobic conditions, so the final product is N₂O rather than N₂. After NO₃^-/NH₄⁺ was added to P. denitrificans R-1 as an additional electron acceptor, the N₂O reduction capacity of P. denitrificans R-1 was greatly improved. The study of Marques et al. (39) showed that the metabolism of NO₃^- produced higher ATP than N₂O reduction process, so it could provide sufficient energy for the process of cell reduction of external N₂O. The addition of NO₃^- can promote the denitrification and the electron transport and the activities of related enzymes. Because the pathway of N₂O reduction is a part of denitrification, the N₂O consumption would be accelerated by adding the NO₃^- in the medium. Ammonium assimilation is more beneficial for the growth and propagation of bacteria and then convenient for the N₂O reduction. The presence of Cu²⁺ can inhibit the N₂O reduction of P. denitrificans R-1. It has been reported (28) that appropriately increasing Cu²⁺ concentration (0–0.05 mg·L⁻¹) can promote the expression of nosZ gene in denitrifiers Pseudomonas stutzeri PCN-1. The high concentration of Cu²⁺ (0.5–5 mg·L⁻¹) would inhibit the denitrification activity and gene expression of the strain, resulting in more N₂O emission. In this experiment, it was found that the N₂O reduction efficiency of P. denitrificans R-1 gradually decreased at the concentration of 10–100 mg·L⁻¹ of Cu²⁺, and whether it would promote the reduction of N₂O at a lower concentration of Cu²⁺ 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 N₂O 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 N₂O reduction process of typical denitrifying bacteria (nosZ-I) not only can consume N₂O produced by themselves but can also consume the external N₂O generated from non-denitrification biological or abiotic pathways under suitable conditions. The exploration of N₂O respiration of P. denitrificans R-1 contributes to further understanding of the regulatory role of microorganisms on N₂O in the natural environment. This is essential for controlling N₂O emissions using microorganisms.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/spectrum.03811-23.

Supplemental material. spectrum.03811-23-s0001.docx.

Fig. S1 and S2; Tables S1 to S6. Fig. S1: N2O reduction process curve of Paracoccus denitrificans R-1 under different electron donors (carbon source). Fig. S2: Effect of different concentrations of inhibitors on the N₂O 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 O₂ concentration. Table S5: Growth of Paracoccus denitrificans R-1 with additional nitrogen sources. Table S6: N₂O consumption rate of Paracoccus denitrificans R-1 in 8 h cultured with different electron donors.

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