Short Communication
Kinetics of Nitrous Oxide (N2O)-reducing Activity of Bradyrhizobium ottawaense by an Automated Analysis
2026 年 41 巻 1 号 論文ID: ME25070
詳細
2026 年 41 巻 1 号 論文ID: ME25070
The biological reduction of N2O, a potent greenhouse gas, is crucial for environmental sustainability. We developed an automated system for continuous N2O monitoring in the gas phase of a flask containing an anaerobic bradyrhizobial culture, and then examined the kinetic parameters of bacterial N2O reduction. The maximum reaction rate (Vmax) was approximately 61-fold higher for Bradyrhizobium ottawaense SG09 (1,471 nmol h–1 109 cells–1) than for B. diazoefficiens USDA110 (24 nmol h–1 109 cells–1). Our kinetics analysis confirmed that SG09 maintained higher N2O-reducing activity than USDA110 even at the atmospheric concentration of N2O (0.34 ppm).
Atmospheric nitrous oxide (N2O) is a major greenhouse gas that contributes to both global warming and ozone layer depletion (Ravishankara et al., 2009; IPCC, 2021). With a global warming potential that is approximately 300-fold that of carbon dioxide (CO2) and a long atmospheric lifetime, N2O exerts long-term effects on Earth’s radiative budget. Agricultural activities—particularly the application of nitrogen fertilizers—are the largest source of anthropogenic N2O emissions (Tian et al., 2020). Therefore, the mitigation of these emissions is an urgent challenge for establishing sustainable agriculture.
N2O is generated via several processes by diverse soil bacteria, fungi, and archaea from nitric oxide (NO) during incomplete denitrification as well as during nitrification (Butterbach-Bahl et al., 2013). Only one bacterial enzyme, N2O reductase (encoded by nosZ), reduces N2O to N2 (Kuypers et al., 2018).
Soybean fields emit N2O from degraded root nodules (Sánchez and Minamisawa, 2019). An inoculation with Bradyrhizobium diazoefficiens, carrying the clade I nosZ gene, was previously shown to reduce N2O emissions from soybean fields (Itakura et al., 2013), which was subsequently confirmed in Japan (Akiyama et al., 2016), France (Hénault et al., 2022), and South America (Obando et al., 2022). Under anaerobic free-living conditions, the N2O-reducing activities of Bradyrhizobium ottawaense were ~5-fold those of B. diazoefficiens USDA110, and were attributed to higher nosZ expression in B. ottawaense (Wasai-Hara et al., 2023).
The ability of N2O-reducing rhizobia to mitigate N2O is crucial for sustainable agriculture. However, to maximize their effectiveness, a quantitative understanding of the reaction kinetics of N2O reductase is essential. A kinetic analysis reveals the Michaelis constant (Km), indicating the enzyme’s affinity for N2O, and the maximum reaction rate (Vmax), showing its maximum reduction capacity. Although these parameters have been reported as important indicators in wastewater treatment and farmland, a kinetic analysis of the N2O-reducing activity of symbiotic (brady)rhizobia has yet to be conducted (Yoon et al., 2016; Suenaga et al., 2018, 2019; Qi et al., 2022; Wang et al., 2023; Hiis et al., 2024).
Conventional GC-based N2O measurements typically rely on a manual gas injection, hindering real-time monitoring capability. While automated systems (e.g., GC or N2O microelectrodes) exist, their high cost or operational complexity limits broad accessibility (Molstad et al., 2007; Suenaga et al., 2018). To address this knowledge gap, we conducted a kinetic analysis of the N2O-reducing activity of soybean bradyrhizobia. We developed an automated, continuous N2O measurement system using a gas chromatograph to evaluate the kinetics of the high N2O-reducing activity of B. ottawaense and the reference N2O-reducing activity of B. diazoefficiens (Wasai-Hara et al., 2023).
The measurement system connects a sample flask to a GS5100S Auto Gas Sampler (sample loop volume 0.3 mL; GL Sciences) and uses a peristaltic pump (AC-2110 II; ATTO Corporation) to circulate the gas phase between them (Fig. 1). The sampler is connected to a gas chromatograph (GC-2014; Shimadzu) equipped with an electron capture detector (ECD). The system automatically and continuously measures N2O and O2 concentrations in the gas phase by injecting the gas circulating in the sample loop into the GC at fixed time intervals (Fig. 1 and S1). The sampler’s cycle consists of “Charge”, “Balance”, “Injection”, and “Interval” steps, with customizable durations and cycle counts. The “Injection” time is the period of time during which the gas from the sample loop flows into the GC for measurement; the peristaltic pump continuously circulates gas from the flask through the sample loop during the other steps.
Diagram of the automated continuous N2O measurement system. The system consists of a sample flask, a peristaltic pump for gas circulation, a sample loop, and a gas chromatograph (GC) equipped with an electron capture detector (ECD). The diagram shows the gas flow path (indicated by arrows) from the headspace of the flask through the sample loop for repeated sampling and injection into the GC, which allows for the real-time monitoring of N2O concentrations. To maintain anaerobic conditions, the system uses a Sampling/Purge Valve (a 4-port, 2-position valve): for sampling through the sample loop, the black lines are used (1–4, 3–2); and for purging the system with N2 gas, the white lines are used (1–2, 3–4). Additionally, the internal valve position of the Auto Gas Sampler changes depending on the measurement cycle: during the “Injection” step, the valve is set to the white lines (1–6, 5–4, 3–2) to inject the sample loop gas into the GC; otherwise, the black lines (1–2, 3–4, 5–6) are used for continuous circulation.
GC calibration was performed using N2O gas concentrations of 10, 50, 100, 500, 1,000, and 4,000 ppm prepared in N2 within cell-free assay flasks. The automated system yielded a linear calibration curve (R2=0.996, n=5) for the quantitative measurement of N2O concentrations throughout the experiment (Fig. S2). Since 0.3 mL of the GC carrier gas (N2) was injected into the flask during each cycle, the measured N2O concentration (Cmeasured) was corrected (Ccorrected) to eliminate this mechanical effect using the formula below.
where Vinjection is 0.3 mL, Vheadspace is the headspace volume of the sample flask, and n is the sampling number.
SG09 and USDA110 were cultured at 30°C for 4–6 days in 200 mL of HM medium (Cole and Elkan, 1973). The cultures were then centrifuged at 9,000×g for 10 min, and the supernatant was discarded. The pellets were resuspended in modified HMM medium (Sameshima-Saito et al., 2004) containing trace metals (0.55 μM Na2MoO4·2H2O, 1 μM FeCl2, and 1 μM CuSO4·2H2O) and L-arabinose (5 g L–1) to OD660 of 0.5 (path length of 10 mm, UV-1200; Shimadzu). Cell numbers were counted using a hemocytometer, and cell numbers per unit of turbidity were approximately 1.9×109 cells mL–1 OD660 for both strains.
The bacterial suspension was transferred to a 200-mL Erlenmeyer flask containing a stir bar: 25 mL of SG09 and 100 mL of USDA110. The flask was then connected to the N2O measurement system and maintained at 30°C in a water bath with constant agitation by a magnetic stirrer (estimated speed: ~1,200 rpm) throughout the measurement period. The system was purged with N2 gas at 200 mL min–1 for 15 min to establish anaerobic conditions. Pure N2O gas (100%) was then added to the gas phase through the gas addition port (Fig. 1) to a final concentration of ~2,000 ppm for SG09 and ~1,000 ppm for USDA110 to allow for the observation of clear kinetic differences within the measurement period. In the SG09 kinetic analysis, the process of adding N2O gas was repeated three times after N2O depletion. A peristaltic pump was used to circulate the gas phase between the flask and the Auto Gas Sampler at 25 mL min–1. The sampler was set with a cycle of “Charge” for 60 s, “Balance” for 10 s, “Injection” for 240 s, and “Interval” for 50 s, allowing for the measurement of N2O and O2 concentrations every 6 min.
By using the corrected N2O concentrations, we calculated the substrate concentration, [S], from the midpoint of two consecutive measurement points and the reaction rate, v, from the slope between them. The relationship between [S] and v was fit to the Michaelis–Menten equation:
using the least squares method in R v. 4.2.2 software (R Core Team, 2022) to assess the Km and maximum reaction rate (Vmax) of N2O-reducing activity (Suenaga et al., 2018, 2019; Wang et al., 2023). Cell-specific Vmax was calculated using the average cell number measured before and after the kinetic analysis. The kinetic analysis was performed for each species in independent triplicate experiments.
To validate the N2O measurement system, we connected a flask containing 25 mL of HM medium without bacteria. After purging the flask with N2, ~1,400 ppm of N2O gas was added. N2O and O2 concentrations were measured at 6-min intervals for 6 h. The N2O concentration gradually decreased with each measurement (Fig. S3). We attributed this to the introduction of 0.3 mL of the GC carrier gas (N2) into the flask per measurement cycle via the sample loop. When the N2O concentration was corrected for this influx using the formula defined above, the decrease in the N2O concentration was almost eliminated (Fig. S3). On the other hand, the O2 concentration was below the detection limit (0.05%) after N2 purging and remained very low (<0.1%) thereafter, confirming that anaerobic conditions were maintained.
We analyzed the N2O-reducing activities of SG09 and USDA110 using the N2O measurement system. After N2O was added to the gas phase of the SG09 culture at ~2,000 ppm, the N2O concentration rapidly decreased to below the detection limit (<5 ppm) in approximately 60 min (Fig. 2A). Subsequent additions of N2O resulted in similar decreases (Fig. 2A). In contrast, when USDA110 was measured at the same cell density as SG09, the addition of N2O at 1,000 ppm decreased to only ~600 ppm after 720 min (Fig. S4). To ensure sufficient kinetics data while avoiding potential density-dependent effects, we maintained cell density, but used 100 mL of a bacterial suspension for the USDA110 analysis. Nevertheless, USDA110 took 200 min to reduce N2O below the detection limit (Fig. 2B).
Kinetic analysis of N2O reduction by Bradyrhizobium ottawaense SG09 and B. diazoefficiens USDA110. (A, B) Changes in N2O consumption over time by (A) SG09 and (B) USDA110, as measured by gas chromatography. (C, D) Michaelis–Menten plots showing the relationship between N2O concentrations and the reaction rates of (C) SG09 and (D) USDA110. Panels show representative results from one of three independent replicate experiments. Data from all three replicates are available in the Supplementary Information as Fig. S5 and Fig. S6.
After the correction of measured N2O concentration data, we calculated [S] and v and fit the data to the Michaelis–Menten equation using the least squares method to assess Km and Vmax (Fig. 2C and D). The Km of SG09 was 227.7 ppm and Vmax was 1,470.9 nmol h–1 109 cells–1. The Km of USDA110 was 68.1 ppm and Vmax was 23.6 nmol h–1 109 cells–1. The Km and Vmax of SG09 were ~3× and ~60× those of USDA110 (Table 1). At the ambient atmospheric concentration of N2O (0.34 ppm), the calculated N2O reduction rate was 2.24 nmol h–1 109 cells–1 for SG09 and 0.12 nmol h–1 109 cells–1 for USDA110. This suggests that SG09 maintained high N2O-reducing activity, even at atmospheric N2O (Table 1). The Km values of both strains were similar to those of nosZ-carrying bacteria in clades I and II, and while the Vmax of SG09 was equivalent, that of USDA110 was markedly low (Table 2).
Estimated Km and Vmax of N2O-reducing activities of Bradyrhizobium ottawaense SG09 and B. diazoefficiens USDA110
| Strain | Replication | Cell number cells flask–1 |
Km ppm N2O |
Vmax | V340 ppb
N2O nmol N2O h–1 109 cells–-1 |
|
|---|---|---|---|---|---|---|
| nM N2O min–1 flask–1 | nmol N2O h–1 109 cells–1 | |||||
| SG09 | Rep_1 | 2.3.E+10 | 189.0 (6.3e-07) | 2540.8 (<2e-16) | 1499.5 | |
| Rep_2 | 2.5.E+10 | 265.3 (1.6e-09) | 2657.7 (<2e-16) | 1414.0 | ||
| Rep_3 | 2.4.E+10 | 228.7 (3.7e-09) | 2610.1 (<2e-16) | 1499.4 | ||
| Average | 227.7±38.2 | 1470.9±49.4 | 2.24±0.44 | |||
| USDA110 | Rep_1 | 1.0.E+11 | 65.5 (6.8e-06) | 318.9 (<2e-16) | 27.5 | |
| Rep_2 | 9.5.E+10 | 68.0 (6.1e-03) | 223.6 (<2e-16) | 24.0 | ||
| Rep_3 | 8.4.E+10 | 70.9 (1.8e-05) | 202.1 (<2e-16) | 19.2 | ||
| Average | 68.1±2.7 | 23.6±4.1 | 0.12±0.03 | |||
Kinetic parameters were assessed by fitting N2O reduction data to the Michaelis–Menten equation. Values represent the means±standard deviation of three independent replicate experiments. Values in parentheses are P-values for the Michaelis–Menten equation fit.
Estimated kinetic parameters of N2O-reducing activities of SG09, USDA110, and previously reported clade I and II nosZ-carrying bacteria
| Strain | nosZ type | Km (μM) | Vmax (mol g DW–1 h–1) | Reference |
|---|---|---|---|---|
| Bradyrhizobium ottawaense SG09 | Clade I | 9.16 | 0.007 | This study |
| Bradyrhizobium diazoefficiens USDA110 | Clade I | 2.72 | 0.0003 | This study |
| Pseudomonas stutzeri JCM5965 | Clade I | 4.01 | 0.008 | Suenaga et al., 2018 |
| Pseudomonas stutzeri DCP-1 | Clade I | 35.5 | 0.250 | Yoon et al., 2016 |
| Shewanella loihica PV-4 | Clade I | 7.07 | 0.027 | Yoon et al., 2016 |
| Paracoccus denitrificans NBRC102528 | Clade I | 34.8 | 0.003 | Suenaga et al., 2018 |
| Cloacibacterium sp. CB-01 | Clade II | 12.9 | 0.006 | Hiis et al., 2024 |
| Dechloromonas aromatica RCB | Clade II | 0.32 | 0.028 | Yoon et al., 2016 |
| Anaeromyxobacter dehalogenans 2CP-C | Clade II | 1.34 | 0.001 | Yoon et al., 2016 |
| Azospira sp. I09 | Clade II | 1.55 | 0.021 | Suenaga et al., 2019 |
| Azospira sp. I13 | Clade II | 2.10 | 0.090 | Suenaga et al., 2019 |
Vmax values cell–1 dry weight of SG09 and USDA110 were calculated using the methodology of Hiis et al. (2024). The Km and Vmax values for other bacteria were cited from the study by Hiis et al. (2024) and its references.
Although N2O microelectrodes enable the real-time measurement of dissolved gas in wastewater (Suenaga et al., 2018, 2019; Qi et al., 2022; Wang et al., 2023), they are expensive, fragile, and unsuitable for crucial gas-phase N2O measurements in soil microbial studies. While Molstad et al. (2007) established a high-throughput multi-sample GC system, we designed a simpler alternative using existing components (Fig. 1). Our system prioritizes a simpler operation and detailed, continuous measurements for a single sample. This simple design allows for the easy measurement of N2O-reducing activity by those with the ability to prepare a bacterial culture. It offers several other advantages: the nitrogen gas purge line facilitates the easy establishment of anaerobic conditions. Its flexibility in changing the gas species and composition of the purge line also enables measurements under aerobic and microaerobic conditions. The system’s modular design also allows for easy adaptation to measurements of other greenhouse gases once the appropriate GC detector and column are used. Furthermore, it is broadly applicable, allowing not only for the sampling of microbial cells for a subsequent gene expression analysis, but also for N2O reduction measurements from a wide range of samples, including bacterial cultures, root nodules, soil, and microbial carriers.
Our kinetic analysis supports previous findings showing that B. ottawaense SG09 exhibited higher N2O-reducing activity than B. diazoefficiens USDA110 (Wasai-Hara et al., 2023). The estimated Vmax of SG09 was 1,470.9 nmol h–1 109 cells–1—within the reported range of 1,350–3,000 nmol h–1 109 cells–1. In contrast, the Vmax of USDA110 was 23.6 nmol h–1 109 cells–1—markedly lower than reported values of 300–500 nmol h–1 109 cells–1. This discrepancy may be attributed to differences in experimental conditions: the present study used cells grown under aerobic conditions, while the previous study used cells precultured overnight under N2O respiration conditions to fully induce N2O reductase activity before measurements (Wasai-Hara et al., 2023). However, the N2O-reducing activity of USDA110 measured under non-induced conditions was ~12 nmol h–1 109 cells–1 (Itakura et al., 2008), similar to the Vmax estimated herein. These results suggest that the kinetic parameters obtained using our novel automated continuous measurement system are reliable and also indicate that SG09 has an intrinsic ability to rapidly express or activate N2O reductase at a high level without the need for pre-induction. This characteristic gives SG09 a marked advantage for N2O mitigation in dynamic soil environments, where N2O levels fluctuate.
SG09 had a higher Km value than USDA110. While a lower Km value indicates a higher affinity for the substrate (N2O), suggesting that USDA110 is more reactive under low N2O concentrations, our calculations showed that the SG09 reaction rate still surpassed that of USDA110 at an atmospheric N2O level (0.34 ppb). Therefore, from the perspective of N2O removal from soils where it is being actively generated, SG09 may be regarded as superior due to its higher reaction rate even at an atmospheric concentration. This result highlights the importance of evaluating both Km and Vmax when assessing the overall N2O mitigation potential of a microbial strain under realistic environmental conditions.
Comparisons of the Km and Vmax values of SG09 and USDA110 with those of previously reported clades I and II nosZ-carrying bacteria showed that Km values were similar. However, the Vmax value of USDA110 was low, whereas that of SG09 was equivalent to that of Cloacibacter sp. CB-01, a strain reported to be effective in reducing farmland N2O emissions (Hiis et al., 2024), suggesting the similar effectiveness of B. ottawaense SG09 (Table 2).
Itakura, M., and Minamisawa, K. (2026) Kinetics of Nitrous Oxide (N2O)-reducing Activity of Bradyrhizobium ottawaense by an Automated Analysis. Microbes Environ 41: ME25070.
https://doi.org/10.1264/jsme2.ME25070
This research was supported by the JPNP18016 project commissioned by the New Energy and Industrial Technology Development Organization (NEDO). We thank Kengo Kubota (Tohoku University) for supporting fitting calculations and for critically reading the manuscript.
Conflicts of interestThe authors declare that there are no conflicts of interest.
