Anoxic growth of Ensifer meliloti 1021 by N2O-reduction, a potential mitigation strategy

Denitrification in agricultural soils is a major source of N₂O. Legume crops enhance N₂O emission by providing N-rich residues, thereby stimulating denitrification, both by free-living denitrifying bacteria and by the symbiont (rhizobium) within the nodules. However, there are limited data concerning N₂O production and consumption by endosymbiotic bacteria associated with legume crops. It has been reported that the alfalfa endosymbiont Ensifer meliloti strain 1021, despite possessing and expressing the complete set of denitrification enzymes, is unable to grow via nitrate respiration under anoxic conditions. In the present study, we have demonstrated by using a robotized incubation system that this bacterium is able to grow through anaerobic respiration of N₂O to N₂. N₂O reductase (N₂OR) activity was not dependent on the presence of nitrogen oxyanions or NO, thus the expression could be induced by oxygen depletion alone. When incubated at pH 6, E. meliloti was unable to reduce N₂O, corroborating previous observations found in both, extracted soil bacteria and Paracoccus denitrificans pure cultures, where expression of functional N₂O reductase is difficult at low pH. Furthermore, the presence in the medium of highly reduced C-substrates, such as butyrate, negatively affected N₂OR activity. The emission of N₂O from soils can be lowered if legumes plants are inoculated with rhizobial strains overexpressing N₂O reductase. This study demonstrates that strains like E. meliloti 1021, which do not produce N₂O but are able to reduce the N₂O emitted by other organisms, could act as even better N₂O sinks.

Introduction

The presence of N₂O in the atmosphere has been known since 1939 (Adel, 1939). However, its importance to the global environment was not recognized until the early 1970s when scientists hypothesized that N₂O released into the atmosphere could activate reactions in the stratosphere that contribute to the depletion of the ozone layer (Crutzen, 1974). The fourth assessment report of the intergovernmental Panel on Climate Change (IPCC, 2007) estimated N₂O emissions from both natural and anthropogenic sources to be 8.5–27.7 Tg N₂O/year. The terrestrial ecosystems are the main source of N₂O, accounting about 65% of total emissions. Agricultural activities are the major sources of N₂O emissions, accounting for 60–80% of the anthropogenic N₂O sources, mostly as N inputs to agricultural soils (Smith, 2008; Smith et al., 2012). These N₂O emissions are likely to increase with the predicted expansion in the use of nitrogenous fertilizers in order to satisfy the escalating demand for food of the growing world population.

A variety of biological pathways are involved in N₂O emissions from soils, and it has been estimated that >65% of the atmospheric N₂O derives from microbial N transformations, mainly through the processes nitrification and denitrification (Thomson et al., 2012). Of these, denitrification is generally considered to be the largest source of N₂O and, depending on the type of microorganisms involved and the environmental conditions, this process can serve not only as source but also as sink for N₂O (Thomson et al., 2012). Denitrification is the respiratory reduction of nitrogen oxides (NOx) which enables facultative aerobic bacteria to survive and multiply under oxygen-limiting conditions. During this process nitrate (NO⁻₃) is converted into molecular nitrogen (N₂) via nitrite (NO⁻₂) and the gaseous intermediates nitric oxide (NO) and nitrous oxide (N₂O) (Zumft, 1997).

In contrast to the variety of N₂O sources in soils, removal of N₂O is only achieved by the last step of the denitrification process which is catalyzed by the N₂O reductase (N₂OR) enzyme encoded by the nosZ gene. Recent reports have demonstrated that diverse microbial taxa possess divergent nos clusters with genes that are related yet evolutionarily distinct from the typical nos genes of denitrifiers (Sanford et al., 2012). In fact, phylogenetic analyses of the nosZ gene revealed two distinct clades of nosZ differing in their signal peptides, indicating differences in the translocation pathway to the N₂OR across the membrane (Jones et al., 2013). The expression and activity of N₂OR is a natural target in the search for options to mitigate N₂O emission from agricultural soils (Richardson et al., 2009). A promising mitigation strategy suggested recently is to stimulate N₂O reductase by sustaining a high soil pH (Bakken et al., 2012). The latter is motivated by recent demonstrations that reduction of N₂O is severely inhibited by suboptimal pH in the model organism Paracoccus denitrificans (Bergaust et al., 2010), in bacterial communities extracted from soils (Liu et al., 2014), and in intact soils (Raut et al., 2012; Qu et al., 2014). Another interesting option would be to alter the composition of the denitrifying community of soils, the objective being to enhance the growth of organisms with high N₂O reductase activity. This would be a daunting task if the free-living soil bacteria were the target, but plant-associated bacteria appear more promising.

Rhizobia is a general term that describes bacteria that have the ability to establish N₂-fixing symbiosis in legume roots or on the stems of some aquatic leguminous plants. In addition to fixing N₂, many rhizobial strains have genes for enzymes of some or all of the four reductase reactions for denitrification. Several studies have reported that legume crops induce N₂O emission by providing N-rich residues for decomposition (Baggs et al., 2000). In addition to soil denitrifiers, endosymbiotic bacteria may be partly responsible for this legume-induced N₂O emission, since most rhizobia are able to denitrify under free-living and under symbiotic conditions (Bedmar et al., 2005; Delgado et al., 2007; Sanchez et al., 2011). Increased N₂O emissions due to degradation of nodules were reported in soybean ecosystems (Inaba et al., 2012). Based on this, Itakura et al. (2013) hypothesized and proved that N₂O emission from soil could be reduced by inoculating soybean plants with a nosZ-overexpressing strain of Bradyrhizobium japonicum. This suggests that root nodules of leguminous plants are net sources or sinks for N₂O. Thus, the investigation of denitrification among rhizobia may provide novel options for reducing N₂O emissions from soils.

Ensifer (formerly Sinorhizobium) meliloti 1021 is a key model organism for studying the symbiotic interaction between rhizobia and plants of the genera Medicago, Melilotus, and Trigonella, that has also been extensively used in previous works to better understand the regulation and symbiotic characterisation of E. meliloti denitrification genes (Bobik et al., 2006; Meilhoc et al., 2010; Horchani et al., 2011). In fact, analysis of the Ensifer meliloti 1021 genome sequence revealed the presence of the napEFDABC, nirK, norECBQD, and nosRZDFYLX denitrification genes encoding a periplasmic nitrate reductase, a copper-containing nitrite reductase, a c-type nitric oxide reductase and a nitrous oxide reductase enzyme, respectively. The involvement of the E. meliloti napA, nirK, norC, and nosZ structural genes in nitrate respiration and in the expression of denitrification enzymes under specific growth conditions (initial oxygen concentrations of 2% and initial cell density of 0.2–0.25) was also demonstrated (Torres et al., 2014). However, this strain has for a long time been considered a partial denitrifier due to its apparent inability to grow under anaerobic conditions with nitrate or nitrite as final electron acceptors (Garcia-Plazaola et al., 1993; Torres et al., 2011a). In order to better understand the truncated denitrification phenotype of E. meliloti 1021, an accurate estimation of the efficiency of the denitrifying process is required. For that purpose, in this work we have used a robotized system which allowed us to simultaneously monitor the O₂ consumption, as well as the consumption and production of each NOx during the transition from oxic to anoxic respiration.

The results convincingly demonstrated that this strain (1021) was unable to reduce NO⁻₃ or NO⁻₂ to N₂O or N₂. In contrast, this bacterium was capable to reduce externally supplied N₂O to N₂, serving as a terminal electron acceptor during anoxic respiration. Thus, our study expands the current understanding of anaerobic respiration in rhizobia and explores the effect of pH, NOx and type of carbon source on N₂O reduction in E. meliloti.

Materials and Methods

Bacterial Strains, and Growth Conditions in Batch Cultures

Ensifer meliloti 1021 (Sm^r, Meade et al., 1982), and napA (napA::mini-Tn5 Sm^r, Km^r, Pobigaylo et al., 2006) and nirK (nirK::mini-Tn5 Sm^r, Km^r, Pobigaylo et al., 2006) mutant strains were used in this study. E. meliloti strains were grown aerobically in 120 mL serum vials containing a triangular magnetic stirring bar and 50 mL of Triptone Yeast (TY) complete medium (Beringer, 1974) at 30°C. All cultures were continuously stirred at 700 rpm to avoid aggregation and ensure complete dispersal of cells. These cultures were then used as inocula into vials containing minimal defined medium (Robertsen et al., 1981) supplemented with or without 10 mM of KNO₃ or 5 mM of NaNO₂. The influence of carbon susbtrates on N₂O uptake capacity was analyzed in minimal medium where the carbon substrate was replaced with either 5 mM of succinate or 5 mM of butyrate as oxidized or reduced carbon sources, respectively. The effect of pH on N₂O uptake capacity was also studied in minimal medium strongly buffered (50 mM phosphate buffer) at pHs 6, 7, and 8. In all the treatments the headspace was filled with an initial concentration of O₂ of 1 or 2% (12 or 24 μM dissolved O₂ at 30°C, respectively). The headspace of experimental vials used to study the N₂O reduction capacity was additionally supplied with an initial concentration of N₂O of 2% (0.42 mM) or 5% (1.2 mM). To avoid possible external contaminations, antibiotics were added to the cultures at the following concentrations (μg mL⁻¹); streptomycin, 200; kanamycin, 200.

Preparation of Incubation Vials

120 mL vials containing 50 mL liquid medium were crimp-sealed with rubber septa (Matriks AS, Norway) and aluminum caps to ensure an airtight system. Oxygen from vials was removed by 6 cycles of air evacuation during 360 s and helium (He) filling during 40 s. Constant stirring (400 rpm) was kept to ensure optimal gas exchange between liquid and headspace. Then, vials were injected with the required concentrations of O₂ and N₂O.

Gas Measurements

After inoculation, cultures, blanks, and gas standards were placed in a thermostatic water incubator containing a serial magnetic stirrer at 30°C, with continuous stirring at 700 rpm, and the gas kinetics were monitored in each vial (2 to 3 h intervals). The gas measurements were performed by monitoring the headspace-concentrations of relevant gases (O₂, CO₂, NO, N₂O, and N₂) by repeated gas sampling through the rubber septa of the incubation vials as described by Molstad et al. (2007). The gas samples were drawn by a peristaltic pump coupled to an autosampler (Agilent GC Sampler 80), and with each sampling an equal volume of He was pumped back into the vials. This secured that the gas pressure was sustained near 1 atm despite repeated sampling, but diluted the headspace atmosphere (with He). This dilution was taken into account when calculating rates of production/consumption for each time increment (Molstad et al., 2007). The sampling system was coupled to a gas chromatograph (GC) (Agilent GC -7890A) with two 30 m × 0.53 mm id columns: a Porous Layer Open Tubular (PLOT) column for separation of CH₄, CO₂ and N₂O, and a Molsieve column for separation of O₂ and N₂ (and Ar, Ne). The GC had three detectors: a flame ionization detector (FID), a thermal conductivity detector (TCD), and an electron capture detector (ECD). N₂O was detected by both the ECD and TCD, thus securing accurate measurements at near-ambient concentrations (ECD, linear range 0–4 or 0–20 ppmv, depending on detector temperature) and linear response for higher concentrations (TCD). NO concentrations were determined by a Chemoluminiscence NOx analyser (Model 200A, Advanced Pollution Instrumentation, San Diego, USA).

OD₆₀₀, Nitrate and Nitrite Measurements

Cell densities (OD₆₀₀), nitrate and nitrite concentrations were measured for each sample. Samples were taken from the liquid phase of the vials throughout the experiment to measure OD₆₀₀ (0.7 mL sample), NO⁻₃(0.1 mL sample), and NO⁻₂ (0.1 mL sample) using sterile syringes. For determination of NO⁻₃, a 10 μL aliquot was injected into a purge vessel with heating jacket and condenser (ASM 03292) containing 1 M HCl and vanadium (III) chloride. Temperature of vessel was controlled by a circulating water bath at 95°C and cold water for the condenser. In addition, a gas bubble/NaOH trap with Teflon sleeve (ASM 04000) was used to avoid the corrosive effects of HCl. Vanadium (III)/HCl converts nitrite and S-nitrosocompounds to NO, which is transported (by N₂) to a chemiluminescence detector Nitric Oxide Analyzer NOA 280i (General Electric). N₂ was continuously bubbled through the reducing agent to maintain an anaerobic environment in the system and to transport the NO through the NO analyzer (Walters et al., 1987). The approximate detection limit was 1 pmol NO, equivalent to 0.1 μM (when injecting 10 μL). For determination of NO⁻₂, a 10 μL subsample was injected into a purge vessel (gas bubble/NaOH trap is not needed) containing acetic acid with 1% vol NaI where NO⁻₂ is converted to NO.

Analyses of Kinetics of Aerobic and Anoxic NO⁻₃, NO⁻₂, or N₂O Respiration

Experimental dataset obtained from the series of incubations were used to determine the kinetics of O₂, NO⁻₃ NO⁻₂, or N₂O respiration and NO, N₂O, and N₂ production in order to provide the most accurate information on E. meliloti physiology during the transition from aerobic to NOx anoxic respiration. O₂ and NO concentration in the liquid, determined as μM and nM, respectively, was estimated taking into account the partial pressure of these gases at headspace, their solubilities and transport coefficients between headspace and liquid. Additionally, O₂ concentration in liquid was estimated respective the O₂ respiration rate for each time increment (see Molstad et al., 2007 for details). N₂O was analyzed as μmol N₂O vial⁻¹, whereas N₂ was determined as cumulative net production of N₂. All data were corrected for dilution rates and losses by gas sampling, and leaks due to gas diffusion through the rubber septa. The concentrations of NO⁻₃ and NO⁻₂ were determined at different times compared to the gas sampling. However, we needed values for NO⁻₂ concentrations at the same time as the gas sampling in order to estimate electron flow rates. For this reason, polynomial functions [f(t)] were fitted to the measured NO⁻₃ and NO⁻₂ concentrations, and used to estimate NO⁻₂ concentration at the time of gas samplings. Graphical presentations for NO⁻₃ and NO⁻₂ concentrations include both measured data points and the polynomial function.

The apparent growth rates based on O₂ consumption (μ_ox), and reduction of any NOx during the anoxic phase (μ_anox) were estimated by regression [ln (V_e−) against time] for the phases with exponentially increasing rates. Yield (cells pmol⁻¹ e⁻) calculation was based on the number of cells rendered per pmol electron used by the respiratory terminal oxidases to reduce O₂ to H₂O during oxic phase (Yield_ox) or by the complete set of denitrifying reductases to reduce NO⁻₃ NO⁻₂ or N₂O to N₂ during anoxic phase (Yield_anox). V_max is an useful parameter that can tell us the efficiency for O₂ and NOx respiration per cell. It estimates the maximal velocities per cell and per hour for the reduction of O₂ and NO_x. This parameter is based on the fmol of electrons used by the terminal oxidases and denitrifying enzymes to reduce O₂ or NOx, respectively, per cell and per hour. For further details regarding these calculations, see Molstad et al. (2007) and Nadeem et al. (2013).

Results

Kinetics of Aerobic Respiration

E. meliloti strain 1021 was grown aerobically for 30 h with vigorous stirring (700 rpm) until a maximal optical density at 600 nm (OD₆₀₀) of ~0.3 to avoid generation of localized anoxic conditions due to cell aggregation. Then, an aliquot was used to inoculate the culture vials to an initial OD₆₀₀ of 0.01 (8 × 10⁶ cells mL⁻¹). The medium contained either 10 mM of nitrate (Figure 1), 5 mM NO⁻₂ (Figure 2) or 10 mM nitrate plus 5% N₂O (1.2 mM N₂O concentration in the liquid when in equilibrium with the headspace) (Figure 3). In all the treatments for studying the kinetics of aerobic respiration, the initial O₂ concentration in the headspace was 2%. Figure 1A shows the measured OD₆₀₀, O₂, NO, N₂O, and N₂ concentrations in the medium for a single vial throughout the 40 h incubation in the presence of nitrate. NO⁻₃ depletion and production of NO⁻₂ is also shown (Figure 1A, insert). In nitrate-treated cells, oxygen was consumed within the first 15 h, OD₆₀₀ increased linearly with the cumulative O₂ consumption (r²) = 0.9877, and remained practically constant throughout the anoxic phase. Rates of O₂ consumption for each time increment between two samplings were used to calculate electron (e⁻) flow rates to oxygen (V_e−O2). As shown in Figure 1B, V_e−O2 increased exponentially throughout the first 7 h in proportion with the increase in OD₆₀₀ (r² = 0.9105), and declined gradually in response to diminishing O₂ concentrations. The initial exponential increase in electron flow during oxic respiration can be taken as an indirect measure of growth rate (μ_ox) (Liu et al., 2013). Thus, the apparent μ_ox estimated by linear regression of ln (V_e−O2) against time was 0.30 (±0.03) h⁻¹ (Figure 1B, Table 1A). The final OD₆₀₀ was 0.15 (±0.02) (1.60 × 10⁸ cells mL⁻¹, Table 1B) resulting in a yield of 24.6 (±2.8) cells pmol⁻¹e⁻ to O₂ (Table 1A). The apparent maximum specific respiration rate, V_max, which is a useful indicator of the respiration per cell, was 11.6 (±0.5) fmol e⁻ cell⁻¹ h⁻¹ for oxygen respiration in cells grown in the presence of nitrate (Table 1A).

FIGURE 1

Figure 1. Kinetics of O₂ and NO⁻₃ respiration. (A) Absorbance at 600 nm (OD₆₀₀), O₂ consumption, NO⁻₃ depletion (insert), and production of NO⁻₂ (insert), NO, N₂O, and N₂ by E. meliloti 1021 when incubated in the presence of 10 mM NO⁻₃ in the medium and an initial O₂ concentration of 2% in the headspace. (B) The electron flow rate to O₂ is shown as log-transformed values for the phases with exponential increase (filled circle symbols). The slopes estimating apparent growth rates were 0.3 (± 0.03) h⁻¹ and 0 for oxic and anoxic phase, respectively. Cultures with an initial OD₆₀₀ of 0.01 were vigorously stirred at 700 rpm. The result shown is for a single vial. Several replicates were analyzed, with similar results, although the exact timing of events was not the same. However, the consistency of the observations is demonstrated in Table 1 where averages of at least three different cultures are reported.

FIGURE 2

Figure 2. Kinetics of O₂ and NO⁻₂ respiration. (A) Absorbance at 600 nm (OD₆₀₀), O₂ consumption, NO⁻₂ depletion (insert), and production of NO, N₂O, and N₂ by E. meliloti 1021 when incubated in the presence of 5 mM NO⁻₂ in the medium and an initial O₂ concentration of2% in the headspace. (B) The electron flow to O₂ shown as log-transformed values for the phases with exponential increase (filled circle symbols). The slopes estimating apparent growth rates were 0.11 (±0.02) h⁻¹ and 0 for oxic and anoxic phase, respectively. Cultures with an initial OD₆₀₀ of 0.01 were vigorously stirred at 700 rpm. The result shown is for a single vial. Several replicates were analyzed, with similar results, although the exact timing of events was not the same. However, the consistency of the observations is demonstrated in Table 1 where averages of at least three different cultures are reported.

FIGURE 3

Figure 3. Kinetics of O₂ and N₂O respiration. (A) Absorbance at 600 nm (OD₆₀₀), O₂ and N₂O depletion, and production of NO, and N₂ by E. meliloti 1021 when incubated in the presence of 10 mM NO⁻₃ in the medium and an initial O₂ concentration of 2% in the headspace plus 5% N₂O. (B) The electron flow to O₂ or to N₂O is shown as log-transformed values for the oxic (filled circle symbols) and anoxic phase (filled square symbols) with exponential increase. The slopes estimating apparent growth rates were 0.28 (±0.03) h⁻¹ for the oxic phase, and 0.11 (±0.03) h⁻¹ for the anoxic phase, respectively. Cultures with an initial OD₆₀₀ of 0.01 were vigorously stirred at 700 rpm. The result shown is for a single vial. Three replicates were analyzed in parallel with similar results. Consistency of the observations are demonstrated in Table 1 where averages of at least three different cultures are reported.

TABLE 1

Table 1. Summary of oxic and anoxic growth parameters (A)¹ Depending on the presence of nitrogen oxides, and the subsequent conversion of the nitrogen oxides present (B)².

O₂ uptake and growth kinetics were also analyzed in cells grown in the presence of 5 mM NO⁻₂ as final electron acceptor (Figure 2). For this treatment, O₂ was consumed within the first 30 h of incubation showing a delay in comparison to NO⁻₃ treatment (Figure 2A). As observed in nitrate-treated cells, OD₆₀₀ also increased during the oxic phase in proportion with O₂ consumption, and remained constant during the anoxic phase. The estimated oxic growth rate in the presence of nitrite (linear regression of ln(V_e−O2) against time was μ_ox = 0.11 (±0.02)h⁻¹ (Figure 2B, Table 1A) and the estimated cell yield was only 14.1 (±1.1) cells pmol⁻¹ e⁻ (Table 1A). The estimated V_max for oxygen respiration in cells grown in the presence of nitrite was 8.2 (±0.7) fmol e⁻ cell⁻¹ h⁻¹ (Table 1A). Thus, the presence of NO⁻₂ in the medium appeared to exert an inhibitory effect on the oxygen respiration by terminal respiratory oxidases, resulting in lower V_max and cell yield per mol electron compared to cells grown in the presence of nitrate.

Finally, kinetics of O₂ respiration were also analyzed when cells were incubated in vials containing minimal medium with 10 mM of NO⁻₃, and an initial concentration of 5% N₂O and 2% O₂ in the headspace. Figure 3A shows the measured O₂, NO, N₂O, and N₂ for a single vial throughout the 40 h incubation, as well as the OD₆₀₀. In this case, oxygen was consumed within the first 15 h and the OD₆₀₀ increased in proportion with the cumulative O₂ consumption and continued increasing throughout the anoxic phase. Electron flow rate to O₂ increased exponentially with an apparent growth rate (μ_ox) = 0.28 (±0.03) h⁻¹ (Figure 3B, Table 1A). Cell yield resulting from O₂ respiration was very similar to that observed in nitrate-treated cells [23.1 (±6.2) cells pmol⁻¹ e⁻ with a V_max of 8.9 (±0.13) fmol e⁻ cell⁻¹ h⁻¹] (Table 1A).

Kinetics of NO⁻₃ and NO⁻₂ Respiration

When cells were cultured with NO⁻₃, there was a very low NO⁻₃ consumption rate as well as very low progressive accumulation of NO⁻₂ throughout the entire anoxic phase (Figure 1A, insert), reaching only ~50 μmol vial⁻¹ (which accounts for 10% of the NO⁻₃-N in the medium). Very low levels of NO were also observed (12.40 ± 2.10 nM) after 40 h incubation (Table 1B, Figure 1A). Production of N₂O in the headspace was insignificant and the fraction of NO⁻₃ reduced to N₂ at the end of the incubation was also extremely low (0.9 ± 0.3 %) (Table 1B, Figure 1A). When NO⁻₂ was used as final electron acceptor, the first detection of NO occurred as the oxygen concentration in the liquid reached ~3 μM (Figure 2A, Table 1B). During the subsequent anoxic phase, NO continued to accumulate, reaching 94.20 ±16.90 nM levels at the end of the incubation period (Table 1B, Figure 2A). Similarly as for nitrate-treated cells (Figure 1A), production of N₂O was undetectable and the total, cumulative production of N₂ from the initially provided NO⁻₂-N was also very low (0.18 ± 0.02 %) (Figure 2A, Table 1B). These data show that E. meliloti 1021 was clearly unable to shift effectively to NO⁻₃ or NO⁻₂ based anaerobic respiration. This inability was also confirmed by the lack of increase in measured OD₆₀₀ throughout the anoxic phase (Figures 1, 2). Thus, the apparent growth rate during either NO⁻₃ or NO⁻₂ anoxic respiration (μ_anox) was zero (Figures 1B, 2B, Table 1A). Similar growth rates were observed by using 1 mM or 500 μM NO⁻₂ as electron acceptor (data not shown). One possible explanation to the lack of efficient reduction of NO⁻₃ and NO⁻₂ could be that rapid depletion of the oxygen in these cultures may have resulted in entrapment of the bacteria in anoxia, as shown previously for P. denitrificans by Bergaust et al. (2010). To test this hypothesis, we performed a follow-up experiment where the stirring speed was reduced from 700 rpm (used in the experiments reported in Figures 1, 2) to 200 rpm, in order to secure a slow transition from oxic to anoxic conditions in the liquid. These cultures showed the same lack of effective transition to denitrification as cultures with vigorous stirring, despite the fact that the cells with low stirring experienced a progressive O₂ limitation during 50 h prior to complete O₂ depletion (see Supplementary Figure S1).

Kinetics of N₂O Respiration

The capacity of E. meliloti 1021 to reduce N₂O was examined in vials containing 10 mM NO⁻₃ in the medium plus 5% N₂O and 2% O₂ initially added to the headspace (Figure 3). As shown in Figure 3A, N₂O was consumed rapidly and N₂ production followed stoichiometrically the reduction of N₂O to its complete depletion (100% of N₂O was converted to N₂ gas) (Figure 3A, Table 1B). As shown in Figure 3A, N₂O reduction was at first detected at an O₂ concentration of 5.9 (±2.6) μM (Table 1B). Traces of NO from NO⁻₃ reduction were also detected (15 ±1.1 nM in the liquid; Table 1B). Final OD₆₀₀ of cells incubated with N₂O was clearly higher than that obtained when cells were incubated only with NO⁻₃ or NO⁻₂ as alternative electron acceptors (Table 1B), demonstrating the capacity of E. meliloti to couple N₂O reduction with growth.

Electron flow to N₂O increased with an apparent growth rate (μ_anox) of 0.11 (±0.03) h⁻¹ estimated by linear regression of ln (V_e−N2O)against time (Figure 3B, Table 1A). Although low rates of electron flow to N₂O occurred after 3 h, it increased sharply after 7 h as the electron flow to oxygen decreased due to oxygen depletion. Thus, the cells were evidently able to shift gradually from respiring O₂ to N₂O, preserving the total electron flow rate essentially unaffected after the depletion of oxygen. As shown in Table 1A, the estimated cell yield from N₂O reduction was 18 (±0.6) cells pmole⁻¹ e⁻. Knowing the yield in cell number per hour and the electron flow rate per hour we could estimate the V_max for N₂O reduction to 5.7 (±1.1) fmol e⁻ cell⁻¹ h⁻¹ (Table 1A).

No_x Molecules Do Not Trigger N₂OR Activity in E. meliloti

To evaluate the effect of NOx molecules as inducers of N₂OR activity, we measured N₂O uptake rates in cultures of E. meliloti 1021 strain that had received 10 mM NO⁻₃ in the medium and compared this with cultures that were not supplemented with NO⁻₃ (Figures 4A,B). The results showed similar N₂O consumption as well as N₂ production rates for the two treatments. Furthermore, no differences in N₂O respiration was found between wild-type cells and strains which were defective in the napA and nirK structural genes when cultured in a medium amended with 10 mM NO⁻₃ (Figures 4A,C,D). The E. meliloti napA or nirK mutants were demonstrated previously to be unable to reduce nitrate and nitrite respectively, to any further NOx intermediary of the denitrification process (Torres et al., 2014). These results suggested that the ability to reduce N₂O was not affected by the presence or absence of NO, NO⁻₂, or NO⁻₃.

FIGURE 4

Figure 4. NOx effect on kinetics of O₂ and N₂O depletion. O₂ and N₂O consumption, and NO and N₂ production by E. meliloti 1021 (A,B), and napA (C) and nirK (D) mutant strains when incubated in the presence of 1% O₂ plus 2% N₂O in the headspace. Cells were incubated in minimal medium with (A,C,D) or without (B) 10 mM NO⁻₃. Cultures with an initial OD₆₀₀of 0.01 were vigorously stirred at 700 rpm. Plotted values are average of three replicate flasks for each treatment, with standard deviation (SD) as vertical bars (n = 3).

Low pH Severely Impaires N₂O Uptake in E. meliloti

Since pH emerges as a master variable controlling the expression of N₂O reductase, in this work we examined the pH effect on the kinetics of N₂O reduction. For that purpose, E. meliloti cells were incubated in minimal medium strongly buffered with phosphate buffer, at pH 6, 7, and 8. Firstly, we grew E. meliloti 1021 cells aerobically to exponential (log) phase at pH 7. Then cells were transferred to the experimental vials containing 5% N₂O and 2% O₂ in the headspace and 10 mM NO⁻₃ in the medium. Rates of O₂ consumption were monitored until depletion and no differences were found between treatments. However, N₂O reduction to N₂ was completely blocked at pH 6 (Figure 5A). Surprisingly, when cells were incubated at pH 8, a significant peak of NO was detected. A negative effect of high pHs on nor expression or Nor activity could explain that transient peak of NO.

FIGURE 5

Figure 5. pH effect on kinetics of O₂ and N₂O depletion. O₂ and N₂O consumption, and NO and N₂production by E. meliloti 1021 when incubated in the presence of 10 mM NO⁻₃ in minimal medium at pH 6 (A), pH 7 (B) and 8 (C), and an initial O₂ concentration of 02% in the headspace plus 5% N₂O. Cultures with an initial OD₆₀₀of 0.01 were vigorously stirred at 700 rpm. Plotted values are average of three replicate flasks for each treatment, with standard deviation (SD) as vertical bars (n = 3). The decline in N₂O concentration at pH = 6 is due to sampling loss, not biological reduction of N₂O to N₂.

Reduced C-sources Attenuates N₂O Uptake in E. meliloti

Carbon availability is another key environmental factor affecting N₂O production in the field. However, information about the implication of specific forms of reductants in N₂O reductase activity is limited. Redox state of the C-sources might influence the amount of electrons available to reduce N₂O to N₂. For that reason, we tested the capacity of E. meliloti 1021 to reduce N₂O in the presence of C-substrates with different redox potential, from highly oxidized as succinate or highly reduced such as butyrate. Aerobically raised cells were collected and inoculated into experimental vials containing minimal medium where glycerol was substituted by either succinate or butyrate. By using the robotized incubation system, rates of O₂ respiration occurring previously to N₂O consumption were also estimated. We found that O₂ respiration from cells incubated in the presence of butyrate was slightly decreased when compared to cells incubated in the presence of succinate (Figures 6A,B). However, rates of N₂O consumption were largely dependent on the oxidized or reduced nature of the carbon source. Thus, when butyrate was used as electron donor, the N₂O reduction to N₂ decreased about 3-fold compared to when succinate was used as the sole carbon substrate (Figures 6A,B).

FIGURE 6

Figure 6. C-source effect on kinetics of O₂ and N₂O depletion. O₂ and N₂O consumption, and NO and N₂ production by E. meliloti 1021 when incubated in the presence of 1% O₂ plus 2 % N₂O in the headspace, and 10 mM NO⁻₃ in Robertsen's medium which contained succinate (A) or butyrate (B) as carbon sources. Cultures with an initial OD₆₀₀ of 0.01 were vigorously stirred at 700 rpm. Plotted values are averages of three replicate flasks for each treatment, with standard deviation (SD) as vertical bars (n = 3).

Discussion

In this work, we have used a robotized incubation system designed to simultaneously monitor with high sensitivity real-time changes in concentrations of O₂, NO⁻₃, NO⁻₂ NO, N₂O, and N₂ during the transition from oxic to anoxic respiration. By using this system, we found that E. meliloti 1021 is unable to reduce NO⁻₃ or NO⁻₂ to N₂O or N₂ during the transition from oxic to anoxic conditions. Consequently, this bacterium was unable to sustain growth during anoxic conditions by using NO⁻₃ or NO⁻₂ as electron acceptors. This is in contrast to recent studies where growth of E. meliloti 1021 was observed during respiration of NO⁻₃ as well as NO⁻₂ (Torres et al., 2011a, 2014). This apparent discrepancy could be due to the different growth conditions and methodological approaches used by Torres et al. (2011a, 2014) and in this work. While they inoculated experimental vials with very high cell density (OD₆₀₀~ 0.2–0.25), which were shaken at 170 rpm, the initial cell density used in the present work was significantly lower (OD₆₀₀~ 0.01), and cultures were stirred at 700 rpm. The reason why we used different conditions in this work is to allow an efficient and controlled gas transfer from the headspace to the liquid and prevented cell aggregation and generation of localized micro-oxic spells during the aerobic phase previous to the transition to anaerobic respiration, as well as accumulation of toxic concentration of metabolites resulting from cell respiration. It might be possible that the growth conditions used by Torres et al. (2011a, 2014) provoked generation of anoxic micro-zones preceding total oxygen depletion due to cell aggregation and consequently the induction of E. meliloti 1021 denitrifying machinery would be facilitated. The present work extends the study of denitrification in E. meliloti by performing an estimation of the growth parameters (i.e., μ, yield, Vmax), as well as a precise quantification of NOx gases dynamics during the transition from oxic to anoxic respiration. This approach, never used in rhizobia, allowed us to perform an accurate estimation of the efficiency of the denitrifying process, and is regarded to be more physiologically relevant than previously conducted growth experiments.

When N₂O was provided as an alternative electron acceptor, anaerobic respiration, and growth was sustained by reducing N₂O to N₂. In this context, a recent report showed the ability of B. japonicum USDA110 to grow anaerobically using exogenous N₂O as the sole electron acceptor (Sanchez et al., 2013). Growth with N₂O as electron acceptor has also been observed in Anaeromyxobacter (Sanford et al., 2012), and in Wolinella, Campylobacter, and Geobacillus (Liu et al., 2008; Kern and Simon, 2009) indicating that the atypical nosZ encodes a functional respiratory terminal N₂O reductase in those bacteria. This is unlike Pseudomonas aeruginosa PAO1, which cannot grow on exogenous N₂O as the only electron acceptor (Bryan et al., 1985; Zumft and Kroneck, 2007).

It is generally considered that low oxygen concentration is a requirement for expression of the denitrification machinery (van Spanning et al., 2007). Especially the N₂OR has been considered as a very O₂ labile reductase which is inactivated by the presence of low amounts of O₂ (Alefounder and Ferguson, 1982; Coyle et al., 1985; Snyder and Hollocher, 1987). In contrast to these observations, our results suggest that expression of N₂OR in E. meliloti might be subjected to a different regulation, in which N₂O reduction occurs even in the presence of oxygen concentrations above 8 μM (Figure 3A).

It has been reported that expression and fine-tuning of the denitrification system also requires the presence of key molecules such as NO⁻₃, NO⁻₂, and NO which, through transcriptional factors and their protein-coupled sensory receptors, act as signals that trigger induction of the denitrification pathway (Zumft and Kroneck, 2007; Spiro, 2012). Our results suggested that oxygen limitation was the only prerequisite for maximal expression of N₂OR in E. meliloti, although we cannot exclude that N₂O is also necessary. The presence of a NOx (NO, NO⁻₂, NO⁻₃) was however not required, since N₂OR activity remained at similar levels in the absence or in the presence of NO⁻₃ in wild-type cells. Furthermore, in cells cultured with NO⁻₃, no differences in N₂OR activity were observed between wild-type, and the napA or nirK mutant strains where the reduction of NO⁻₃ or NO⁻₂ is blocked, respectively. In fact, previous studies of gene expression proposed that limited oxygen tension alone resulted in induction of the expression of the whole nos operon in E. meliloti (Bobik et al., 2006). In contrast to these findings, transcriptional profile analysis suggested that induction of nosR and nosZ gene expression also requires the presence of nitric oxide (Meilhoc et al., 2010). In line with this, recent studies using qRT-PCR showed that maximal transcription of the E. meliloti nosZ gene occurred when cells were subjected to anoxic conditions in the presence of nitrate (Torres et al., 2014). Similarly to our observations, it was recently reported that P. denitrificans is fully able to reduce N₂O in the absence of oxyanions and NO (Bergaust et al., 2012). In contrast, it was proposed that the inability of Pseudomonas aeruginosa PAO1 and Bacillus vireti to grow on exogenous N₂O as the only electron acceptor was because these organisms need NO as an inducer of nosZ transcription (Arai et al., 2003).

Our results clearly showed that E. meliloti 1021 was unable to express N₂OR activity at pH 6. This difficulty in expressing N₂OR at low pH was observed in P. denitrificans (Bergaust et al., 2010) and in suspensions of extracted soil bacteria (Liu et al., 2014). The phenomenon is ecologically important since there is ample evidence that low soil pH results in high N₂O/N₂ product ratios of denitrification (Raut et al., 2012; Qu et al., 2014).

Among the environmental factors that influence N₂O emissions, and specifically the bacterial N₂OR performance, very little is known about the mode in which availability and redox state of C-sources contribute. In this work, the observed attenuated N₂OR activity in the presence of highly reduced C-sources could be attributed to a reduced capacity of cells to metabolize more complex C-substrates such as butyrate, causing a lowered electron flow through the respiratory chain, resulting in a reduced electron availability to reduce N₂O to N₂ by the N₂OR (Morley and Baggs, 2011). Alternatively, a reduced efficiency to metabolize butyrate could be due to the fact that its uptake into cell probably requires active transport, and consequently cells may be subjected to periods of reduced N₂OR activity (Schalk-otte et al., 2000). Supporting this hypothesis, it was found that N₂OR activity was stimulated in the presence of artificial root exudates with easily metabolized C-sources such as glucose, as well as in soils amended with carbohydrates as glucose and starch (Murray et al., 2004; Henry et al., 2008). In addition, a regulatory control on nos transcription could also explain the dependence of the N₂OR activity on the redox state of C-sources. In accordance with this, it was recently reported that expression levels of the B. japonicum NorC component of the nitric oxide reductase in wild-type cells, incubated in minimal medium with succinate as the sole C-source, were significantly higher than those observed in cells incubated in the presence of butyrate (Torres et al., 2011b). Similarly, expression of the B. japonicum fixNOQP genes, encoding the high affinity terminal oxidase cbb₃, decreased when butyrate was the sole carbon source compared to when malate was used (Bueno et al., 2009).

Taken together, these results showed a novel denitrifying phenotype in E. meliloti 1021, for which the reduction of NO⁻₃, or NO⁻₂ was severely impaired, while N₂O was actively reduced. We further demonstrated that the reduction of N₂O sustained growth by E. meliloti 1021. To our knowledge this is the first time that it was demonstrated the capacity of E. meliloti to sustain anoxic respiration by using N₂O as terminal electron acceptor. Since the effect of pH or C-sources on N₂O reductase activity has never been examined in rhizobia, the relevance of this study is to demonstrate that both environmental factors affect N₂O reductase activity in the model alfalfa endosymbiont, E. meliloti 1021. Although this strain is a model organism and is not commercially used as inoculant for alfalfa, the results obtained here could be expanded to more competitive and efficient N₂-fixers inoculants in order to develop strategies to reduce N₂O emissions from alfalfa crops. In fact, despite the large research efforts invested in flux measurement of N₂O emissions, progress in developing efficient mitigation options has hitherto been slow. An essential objective should be to understand the underlying mechanisms and factors that affect the regulation of N₂O consumption and production, and consequently to improve the product stoichiometry of denitrification (N₂O/N₂O + N₂) in terrestrial ecosystems.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

This work was supported by a Fondo Europeo de Desarrollo Regional (FEDER)-co-financed grants (AGL2010-18607 and AGL2013-45087-R) from the Ministerio de Economía y Competitividad (Spain). Grant AGR-1968 and support from the Junta de Andalucía to Group BIO-275 are also acknowledged. We thank A. Becker for providing the E. meliloti mutants. EB was supported by a Personal visiting researcher grant – IS-MOBIL (Oslo University, Norway) and from the Consejo Superior de Investigaciones Cientificas JAE-DOC Programme co-financed by European Social Fund (ESF).

Supplementary Material

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.2015.00537/abstract

Supplementary Figure S1. Kinetics of O₂ depletion and N₂O, NO, and N₂ production. E. meliloti 1021 was incubated in the presence of 10 mM NO⁻₃ in minimal medium and an initial O₂ concentration of 2% in the headspace. Cultures with an initial OD₆₀₀ of 0.01 were vigorously stirred at 200 rpm. Plotted values are averages of three replicate flasks for each treatment, with standard deviation (SD) as vertical bars (n = 3).

References

Adel, A. (1939). Note on the atmospheric oxides of nitrogen. Astrophys. J. 90, 627. doi: 10.1086/144129

PubMed Abstract | CrossRef Full Text | Google Scholar

Alefounder, P. R., and Ferguson, S. J. (1982). Electron transport-linked nitrous oxide synthesis and reduction by Paracoccus dentirificans monitored with an electrode. Biochem. Biophys. Res. Comm. 104, 1149–1155. doi: 10.1016/0006-291X(82)91370-5

PubMed Abstract | CrossRef Full Text | Google Scholar

Arai, H., Mizutani, M., and Igarashi, Y. (2003). Transcriptional regulation of the nos genes for nitrous oxide reductase in Pseudomonas aeruginosa. Microbiology 149, 29–36. doi: 10.1099/mic.0.25936-0

PubMed Abstract | CrossRef Full Text | Google Scholar

Baggs, E. M., Rees, R. M., Smith, K. A., and Vinten, A. J. A. (2000). Nitrous oxide emission from soils after incorporation of crop residues. Soil Use Mgmt. 16, 82–87. doi: 10.1111/j.1475-2743.2000.tb00179.x

CrossRef Full Text | Google Scholar

Bakken, L. R., Bergaust, L., Liu, B., and Frostegård, Å. (2012). Regulation of denitrification at the cellular level a clue to understanding of N₂O emissions from soils. Philos. Trans. R Soc. Lond. B Biol. Sci. 367, 1226–1234. doi: 10.1098/rstb.2011.0321

PubMed Abstract | CrossRef Full Text | Google Scholar

Bedmar, E. J., Robles, E. F., and Delgado, M. J. (2005). The complete denitrification pathway of the symbiotic, nitrogen-fixing bacterium Bradyrhizobium japonicum. Biochem. Soc. Trans. 33, 141–144. doi: 10.1042/BST0330141

PubMed Abstract | CrossRef Full Text | Google Scholar

Bergaust, L., Mao, Y., Bakken, L. R., and Frosteård, Å. (2010). Denitrification response patterns during the transition to anoxic respiration and posttranscriptional effects of suboptimal pH on nitrogen oxide reductase in Paracoccus denitrificans. Appl. Environ. Microb. 76, 6387–6396. doi: 10.1128/AEM.00608-10

PubMed Abstract | CrossRef Full Text | Google Scholar

Bergaust, L., van Spanning, R., Frostegard, Å., and Bakken, L. R. (2012). Expression of nitrous oxide reductase in Paracoccus denitrificans is regulated by oxygen and nitric oxide through FnrP and NNR. Microbiology 158, 826–834. doi: 10.1099/mic.0.054148-0

PubMed Abstract | CrossRef Full Text

Beringer, J. E. (1974). R factor transfer in Rhizobium leguminosarum. J. Gen. Microbiol. 84, 188–198. doi: 10.1099/00221287-84-1-188

PubMed Abstract | CrossRef Full Text | Google Scholar

Bobik, C., Meilhoc, E., and Batut, J. (2006). FixJ: a major regulator of the oxygen limitation response and late symbiotic functions of Sinorhizobium meliloti. J. Bacteriol. 188, 4890–4902. doi: 10.1128/JB.00251-06

PubMed Abstract | CrossRef Full Text | Google Scholar

Bryan, B. A., Jeter, R. M., and Carlson, C. A. (1985). Inability of Pseudomonas stutzeri denitrification mutants with the phenotype of Pseudomonas aeruginosa to grow in nitrous oxide. Appl. Environ. Microbiol. 50, 1301–1303.

PubMed Abstract | Google Scholar

Bueno, E., Richardson, D. J., Bedmar, E. J., and Delgado, M. J. (2009). Expression of Bradyrhizobium japonicum cbb₃ terminal oxidase under denitrifying conditions is subjected to redox control. FEMS Microbiol. Lett. 1, 20–28. doi: 10.1111/j.1574-6968.2009.01711.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Coyle, C. L., Zumft, W. G., Kroneck, P. M. H., Körner, H., and Jakob, W. (1985). Nitrous oxide reductase from denitrifying Pseudomonas perfectomarina: purification and properties of a novel multicopper enzyme. Eur. J. Biochem. 153, 459–467. doi: 10.1111/j.1432-1033.1985.tb09324.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Crutzen, P. J. (1974). Estimates of possible variations in total ozone due to natural causes and human activities. Ambio 3, 201–210.

Google Scholar

Delgado, M. J., Casella, S., and Bedmar, E. J. (2007). “Denitrification in rhizobia-legume symbiosis,” in Biology of the Nitrogen Cycle, eds H. Bothe, S. J. Ferguson, and W. E. Newton (Amsterdam: Elservier Science), 57–66.

Garcia-Plazaola, J. I., Becerril, J. M., Arrese-Igor, C., Gonzalez-Murua, C., and Aparicio-Tejo, P. M. (1993). The contribution of Rhizobium meliloti to soil denitrification. Plant Soil. 157, 207–213. doi: 10.1007/BF00011049

CrossRef Full Text | Google Scholar

Henry, S., Texier, S., Hallet, S., Bru, D., Dambreville, C., Cheneby, D., et al. (2008). Disentangling the rhizosphere effect on nitrate reducers and denitrifiers: insight into the role of root exudates. Environ. Microbiol. 10, 3082–3092. doi: 10.1111/j.1462-2920.2008.01599.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Horchani, F., Prevot, M., Boscari, A., Evangelisti, E., Meilhoc, E., Bruand, C., et al. (2011). Both plant and bacterial nitrate reductases contribute to nitric oxide production in Medicago truncatula nitrogen-fixing nodules. Plant Physiol. 155, 1023–1036. doi: 10.1104/pp.110.166140

PubMed Abstract | CrossRef Full Text | Google Scholar

Inaba, S., Ikenishi, F., Itakura, M., Kikuchi, M., Eda, S., Chiba, N., et al. (2012). N₂O emission from degraded soybean nodules depends on denitrification by Bradyrhizobium japonicum and other microbes in the rhizosphere. Microbes Environ. 27, 470–476. doi: 10.1264/jsme2.ME12100

PubMed Abstract | CrossRef Full Text | Google Scholar

IPCC. (2007). Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change, 2007. Cambridge: Cambridge University Press.

Itakura, M., Uchida, Y., Akiyama, H., Takada, Y., Shimomura, Y., Morimoto, S., et al. (2013). Mitigation of nitrous oxide emissions from soils by Bradyrhizobium japonicum inoculation. Nat. Clim. Change. 3, 208–212. doi: 10.1038/nclimate1734

CrossRef Full Text | Google Scholar

Jones, C. M., Graf, D. R., Bru, D., Philippot, L., and Hallin, S. (2013). The unaccounted yet abundant nitrous oxide-reducing microbial community: a potential nitrous oxide sink. ISME J. 7, 417–426. doi: 10.1038/ismej.2012.125

PubMed Abstract | CrossRef Full Text | Google Scholar

Kern, M., and Simon, J. (2009). Electron transport chains and bioenergetics of respiratory nitrogen metabolism in Wolinella succinogenes and other Epsilonproteobacteria. Biochim. Biophys. Acta 1787, 646–656. doi: 10.1016/j.bbabio.2008.12.010

PubMed Abstract | CrossRef Full Text | Google Scholar

Liu, B., Frostegård, Å., and Bakken, L. R. (2014). Impaired reduction of N₂O to N₂ in acid soil is due to a post transcriptional interference with the expression of nosZ. MBio 5, e01383–e01314. doi: 10.1128/mBio.01383-14

PubMed Abstract | CrossRef Full Text | Google Scholar

Liu, B., Mao, Y., Bergaust, L., Bakken, L. R., and Frostegård, Å. (2013). Strains in the genus Thauera exhibit remarkably different denitrification regulatory phenotypes. Environ. Microbiol. 15, 2816–2828. doi: 10.1111/1462-2920.12142

PubMed Abstract | CrossRef Full Text | Google Scholar

Liu, X., Gao, C., Zhang, A., Jin, P., Wang, L., and Feng, L. (2008). The nos gene cluster from gram-positive bacterium Geobacillus thermodenitrificans NG80-2 and functional characterization of the recombinant NosZ. FEMS Microbiol. Lett. 289, 46–52. doi: 10.1111/j.1574-6968.2008.01362.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Meade, H. M., Long, S. R., Ruvkun, G. B., Brown, S. E., and Ausubel, F. M. (1982). Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J. Bacteriol. 149, 114–122.

PubMed Abstract | Google Scholar

Meilhoc, E., Cam, Y., Skapski, A., and Bruand, C. (2010). The response to nitric oxide of the nitrogen-fixing symbiont Sinorhizobium meliloti. Mol. Plant Microbe Interact. 23, 748–759. doi: 10.1094/MPMI-23-6-0748

PubMed Abstract | CrossRef Full Text | Google Scholar

Molstad, L., Dörsch, P., and Bakken, L. R. (2007). Robotized incubation system for monitoring gases (O₂, NO, N₂O N₂) in denitrifying cultures. J. Microbiol. Methods 71, 202–211. doi: 10.1016/j.mimet.2007.08.011

PubMed Abstract | CrossRef Full Text | Google Scholar

Morley, N., and Baggs, E. M. (2011). Carbon and oxygen controls on N₂O and N₂ production during nitrate reduction. Soil Biol. Biochem. 42, 1864–1871. doi: 10.1016/j.soilbio.2010.07.008

CrossRef Full Text

Murray, P. J., Hatcha, D. J., Dixona, E. R., Stevensb, R. J., Laughlinc, R. J., and Jarvisa, S. C. (2004). Denitrification potential in a grassland subsoil: effect of carbon substrates. Soil Biol. Biochem. 36, 545–547. doi: 10.1016/j.soilbio.2003.10.020

CrossRef Full Text | Google Scholar

Nadeem, S., Dörsch, P., and Bakken, L. R. (2013). The significance of early accumulation of nanomolar concentrations of NO as an inducer of denitrification. FEMS Microbiol. Ecol. 83, 672–684. doi: 10.1111/1574-6941.12024

PubMed Abstract | CrossRef Full Text | Google Scholar

Pobigaylo, N., Wetter, D., Szymczak, S., Schiller, U., Kurtz, S., Meyer, F., et al. (2006). Construction of a large signature-tagged mini-Tn5 transposon library and its application to mutagenesis of Sinorhizobium meliloti. Appl. Environ. Microbiol. 72, 4329–4337. doi: 10.1128/AEM.03072-05

PubMed Abstract | CrossRef Full Text | Google Scholar

Qu, Z., Wang, J., Almøy, T., and Bakken, L. R. (2014). Excessive use of nitrogen in Chinese agriculture results in high N₂O/(N₂O+N₂) product ratio of denitrification, primarily due to acidification of the soils. Glob. Change Biol. 20, 1685–1698. doi: 10.1111/gcb.12461

PubMed Abstract | CrossRef Full Text | Google Scholar

Raut, N., Dörsch, P., Sitaula, B., and Bakken, L. R. (2012). Soil acidification by intensified crop production in South East Asia results in higher N₂O/(N₂+N₂O) product ratio of denitrification. Soil Biol. Biochem. 55, 104–112. doi: 10.1016/j.soilbio.2012.06.011

CrossRef Full Text | Google Scholar

Richardson, D., Felgate, H., Watmought, N., Thomson, A., and Baggs, E. (2009). Mitigating release of the potent greenhouse gas N₂O from the nitrogen cycle–could enzymatic regulation hold the key? Trends Biotechnol. 27, 388–397. doi: 10.1016/j.tibtech.2009.03.009

PubMed Abstract | CrossRef Full Text | Google Scholar

Robertsen, B. K., Aman, P., Darvill, A. G., McNeil, M., and Albersheim, P. (1981). Host-symbiont interactions. V. The structure of acidic extracellular polysaccharides secreted by Rhizobium leguminosarum and Rhizobium trifolii. Plant. Physiol. 67, 389–400. doi: 10.1104/pp.67.3.389

PubMed Abstract | CrossRef Full Text | Google Scholar

Sanchez, C., Bedmar, E. J., and Delgado, M. J. (2011). “Denitrification in Legume-associated endosymbiotic Bacteria,” in Nitrogen Cycling in Bacteria, ed J. W. B. Moir (Norfolk, VA: Caister Academic Press), 197–210.

Google Scholar

Sanchez, C., Itakura, M., Mitsui, H., and Minamisawa, K. (2013). Linked expressions of nap and nos genes in a Bradyrhizobium japonicum mutant with increased N₂O reductase activity. Appl. Environ. Microbiol. 79, 4178–4180. doi: 10.1128/AEM.00703-13

PubMed Abstract | CrossRef Full Text | Google Scholar

Sanford, R. A., Wagner, D. D., Wu, Q., Chee-Sanford, J. C., Thomas, S. H., Cruz-García, C., et al. (2012). Unexpected nondenitrifier nitrous oxide reductase gene diversity and abundance in soils. Proc. Natl. Acad. Sci. U.S.A. 109, 19709–19714. doi: 10.1073/pnas.1211238109

PubMed Abstract | CrossRef Full Text | Google Scholar

Schalk-otte, S., Seviour, R. J., Kuenen, J. G., and Jetten, M. S. M. (2000). Nitrous oxide (N₂O) production by Alcaligenes faecalis during feast and famine regimes. Water Res. 7, 2080–2088. doi: 10.1016/S0043-1354(99)00374-7

CrossRef Full Text | Google Scholar

Smith, K. A., Mosier, A. R., Crutzen, P. J., and Winiwarter, W. (2012). The role of N₂O derived from biofuels, and from agriculture in general, in Earth's climate. Philos. Trans. R Soc. Lond. B Biol. Sci. 367, 1169–1174. doi: 10.1098/rstb.2011.0313

PubMed Abstract | CrossRef Full Text | Google Scholar

Smith, P. (2008). Greenhouse gas mitigation in agriculture. Philos. Trans. R Soc. Lond. B Biol. Sci. 363, 789–813. doi: 10.1098/rstb.2007.2184

PubMed Abstract | CrossRef Full Text | Google Scholar

Snyder, S. W., and Hollocher, T. C. (1987). Purification and some characteristics of nitrous oxide reductase from Paracoccus denitrifcans. J. Biol. Chem. 262, 6515–6525.

PubMed Abstract | Google Scholar

Spiro, S. (2012). Nitrous oxide production and consumption: regulation of gene expression by gas-sensitive transcription factors. Philos. Trans. R Soc. Lond. B Biol. Sci. 367, 1213–1225. doi: 10.1098/rstb.2011.0309

PubMed Abstract | CrossRef Full Text | Google Scholar

Thomson, A. J., Giannopoulos, G., Pretty, J., Baggs, E. M., and Richardson, D. J. (2012). Biological sources and sinks of nitrous oxide and strategies to mitigate emissions. Philos. Trans. R Soc. Lond. B Biol. Sci. 367, 1157–1168. doi: 10.1098/rstb.2011.0415

PubMed Abstract | CrossRef Full Text | Google Scholar

Torres, M. J., Bueno, E., Mesa, S., Bedmar, E. J., and Delgado, M. J. (2011b). Emerging complexity in the denitrification regulatory network of Bradyrhizobium japonicum. Biochem. Soc. Trans. 39, 284–288. doi: 10.1042/BST0390284

PubMed Abstract | CrossRef Full Text | Google Scholar

Torres, M. J., Rubia, M. I., Bedmar, E. J., and Delgado, M. J. (2011a). Denitrification in Sinorhizobium meliloti. Biochem. Soc. Trans. 39, 1886–1889. doi: 10.1042/BST20110733

PubMed Abstract | CrossRef Full Text | Google Scholar

Torres, M. J., Rubia, M. I., de la Peña, T. C., Pueyo, J. J., Bedmar, E. J., and Delgado, M. J. (2014). Genetic basis for denitrification in Ensifer meliloti. BMC Microbiol. 14, 142–151. doi: 10.1186/1471-2180-14-142

PubMed Abstract | CrossRef Full Text | Google Scholar

van Spanning, R. J., Richardson, D. J., and Ferguson, S. J. (2007). “Introduction to the biochemistry and molecular biology of denitrification,” in Biology of the Nitrogen Cycle, eds H. Bothe, S. J. Ferguson, and W. E. Newton (Amsterdam: Elservier Science), 3–20. doi: 10.1016/B978-044452857-5.50002-3

CrossRef Full Text | Google Scholar

Walters, C. L., Gillatt, P. N., Palmer, R. C., and Smith, P. L. (1987). A rapid method for the determination of nitrate and nitrite by chemiluminescence. Food Addit. Contam. 4, 133–140. doi: 10.1080/02652038709373624

PubMed Abstract | CrossRef Full Text | Google Scholar

Zumft, W. G. (1997). Cell biology and molecular basis of denitrification. Microbiol. Mol. Biol. Rev. 61, 533–616.

PubMed Abstract | Google Scholar

Zumft, W. G., and Kroneck, P. M. H. (2007). Respiratory transformation of nitrous oxide (N₂O) to dinitrogen by bacteria and archaea. Ad. in Microb. Physiol. 52, 107–227. doi: 10.1016/S0065-2911(06)52003-X

CrossRef Full Text