Temperature-Related N₂O Emission and Emission Potential of Freshwater Sediment

Abstract

Nitrous oxide (N₂O) is a major radiative forcing and stratospheric ozone-depleting gas. Among natural sources, freshwater ecosystems are significant contributors to N₂O. Although temperature is a key factor determining the N₂O emissions, the respective effects of temperature on emitted and dissolved N₂O in the water column of freshwater ecosystems remain unclear. In this study, 48 h incubation experiments were performed at three different temperatures; 15 °C, 25 °C, and 35 °C. For each sample, N₂O emission, dissolved N₂O in the overlying water and denitrification rates were measured, and N₂O-related functional genes were quantified at regular intervals. The highest N₂O emission was observed at an incubation of 35 °C, which was 1.5 to 2.1 factors higher than samples incubated at 25 °C and 15 °C. However, the highest level of dissolved N₂O and estimated exchange flux of N₂O were both observed at 25 °C and were both approximately 2 factors higher than those at 35 °C and 15 °C. The denitrification rates increased significantly during the incubation period, and samples at 25 °C and 35 °C exhibited much greater rates than those at 15 °C, which is in agreement with the N₂O emission of the three incubation temperatures. The NO₃⁻ decreased in relation to the increase of N₂O emissions, which confirms the dominant role of denitrification in N₂O generation. Indeed, the nirK type denitrifier, which constitutes part of the denitrification process, dominated the nirS type involved in N₂O generation, and the nosZ II type N₂O reducer was more abundant than the nosZ I type. The results of the current study indicate that higher temperatures (35 °C) result in higher N₂O emissions, but incubation at moderate temperatures (25 °C) causes higher levels of dissolved N₂O, which represent a potential source of N₂O emissions from freshwater ecosystems.

1. Introduction

Nitrous oxide (N₂O) constitutes a significant source of global greenhouse gases [1,2], and it plays a major role in ozone depletion in the stratosphere [3]. Therefore, knowledge of the production and emission of N₂O is of great use for scientists to further understand the processes of global warming and the destruction of the stratospheric ozone layer [4]. N₂O is produced by multiple biological pathways, including nitrification, denitrification, and dissimilatory nitrate reduction to ammonium [5,6]. Nitrification is generally the main N₂O source under oxic conditions in soil [7], while denitrification is the main source in the anaerobic environment [8]. Due to different irrigation patterns, such as alternate wetting and drying (AWD) and continuous flooding (CF), the c showed diverse results. In AWD irrigation, the peak of N₂O emission occurred both during the dry and c period. While the emission peak occurred only after fertilizer application in CF conditions. [9]. Because of the aerobic and anaerobic alternation provided by AWD irrigation, nitrification and denitrification were enhanced. The substrate for microbial activity was provided by fertilization. Both of them made a high N₂O emission. However, the continuously anaerobic condition of CF was not favorable for N₂O emission [10]. Because of the high content of organic matter and the anaerobic environment of the sediment [11], the N₂O emissions in aquatic systems are generally much greater than those in soil. This is the result of denitrification processes which greatly dominate over nitrification processes in the generation of N₂O.

Freshwater ecosystems currently produce about 1.8 Tg N-N₂O per year and account for about 25% of global N₂O emissions [12]. The N₂O emissions from freshwater ecosystems are influenced by a variety of environmental factors, such as temperature, pH, dissolved oxygen (DO) and nitrogen concentration in the sediment [13,14,15]. Among them, temperature has been demonstrated to greatly influence N₂O emissions [16,17]. Most studies suggest that higher temperatures increase microbial activity, which leads to increased N₂O release. For example, the N₂O emissions in aquatic ecosystems are normally higher in summer than in colder periods of the year [18,19]. Additionally, freshwater lakes with similar annual temperatures have been shown to have comparable N₂O emission rates, while these rates were higher for lakes exposed to lower temperatures [20]. With the recent findings concerning N₂O-reducing processes and microorganisms, the quest to elucidate the ways by which N₂O emissions are affected has become ever more complicated [21]. It has been observed that increased temperatures promote the greater activity of specific microbes able to reduce N₂O to N₂, thereby decreasing N₂O emissions [22]. On the contrary, a study has reported that N₂O emissions do not respond to variations in temperature [23]. These inconsistent results on the relation between temperature and N₂O emissions could be ascribed to the complicated environmental factors in situ conditions, differing methodologies, such as differences in N₂O gas collection, or the fact that the dominant microbial process of N₂O generation and reduction varied in the studied habitats [24].

To identify the relationship between temperature and N₂O emissions, sediment samples from a freshwater lake located in Guangzhou, China, were collected for incubation experiments at three different temperatures; 15 °C, 25 °C and 35 °C. It was hypothesized that the high N₂O might occur at a higher temperature because of the high microbial activity. Both N₂O emissions and the level of dissolved N₂O in the water column were collected in a time series. By measuring the N₂O concentration, denitrification activity and N₂O-related gene abundance, the current study aims to (i) show the response of N₂O emissions and dissolved N₂O to different temperatures; and (ii) elucidate the microbial background underlying these variations in N₂O characteristics.

2. Materials and Methods

2.1. Experimental Set Up

Sediment and overlying water material were collected in parallel in May 2022 at a waterbody in Guangzhou, China, to be used for incubation experiments which lasted for 48 h. The annual mean temperature at the site ranges between 18–26 °C. The sediment material was incubated in 10 L incubators (POMEX, Beijing, China), and the collected overlying water was hereafter added to a ratio of 3:4 (v/v). The incubations were run at ambient temperatures of 15 °C, 25 °C, and 35 °C controlled by the temperature-controlled incubators. The first sediment and water samples with three replications were taken 12 h after the onset of the incubation experiment to allow ample time for the microbes to acclimatize to the new temperature. Thereafter, sampling took place in 4 h intervals until the 36 h. A final sampling was made at the 48 h.

2.2. Physicochemical Analysis

Sediment ammonium (NH₄⁺), nitrite (NO₂⁻), and nitrate (NO₃⁻) were extracted from 2 g of fresh sediment with 10 mL of 2 M KCl (1:5 wt./vol). The supernatant was filtered through a 0.22 μm membrane filter (Jinlong, Tianjin, China) and determined via a spectrophotometric detection assay [25].

2.3. Calculating N₂O Exchange Flux

The gas exchange flux at the water-gas interface is calculated using the following equation based on the dissolved N₂O:

$$\mathrm{F}=\mathrm{k}\times \left({\mathrm{C}}_{\mathrm{obs}}-{\mathrm{C}}_{\mathrm{eq}}\right)$$

(1)

where F (nmol/m²·h) is the water-air exchange flux. k (cm·h⁻¹) is the gas exchange rate. C_obs is the measured concentration of dissolved N₂O as mentioned above, and C_eq (nmol·L⁻¹) is the concentration of N₂O in the surface water at equilibrium with the atmosphere, which can be calculated using the following equation [26]:

$$\begin{gathered} \mathrm{LnF}={\mathrm{A}}_1+{\mathrm{A}}_2\left(100/\mathrm{T}\right)+{\mathrm{A}}_3\times \mathrm{Ln}\left(\mathrm{T}/100\right)+{\mathrm{A}}_4\times {\left(\mathrm{T}/100\right)}^2+\mathrm{S}\times \left[{\mathrm{B}}_1+ \\ {\mathrm{B}}_2\times \left(\mathrm{T}/100\right)+{\mathrm{B}}_3\times {\left(\mathrm{T}/100\right)}^2\right] \end{gathered}$$

$${\mathrm{C}}_{\mathrm{eq}}=\mathrm{F}\times {\mathrm{C}}_{{\mathrm{N}}_2\mathrm{O}\mathrm{in}\mathrm{atmosphere}}\times {10}^9$$

(2)

where F is experiment value in (mol/L·atm), A₁ = −165.8806, A₂ = 222.8743, A₃ = 92.0792, A₄ = −1.48425, B₁ = −0.056235, B₂ = 0.031619, B₃ = −0.0048472. It is assumed that the concentration of N₂O in the atmosphere is 325 × 10⁻⁹.

The gas exchange rate k (cm·h⁻¹) is measured by the gas tracer method according to the Wanninkhof formula model can accurately estimate the gas exchange rate at different wind speeds [27].

$$\mathrm{k}=0.31\times {\mathrm{U}}_{10}{}^2\times {\left(\frac{{\mathrm{S}}_{\mathrm{c}}}{660}\right)}^{\frac{1}{2}}$$

(3)

where U₁₀ is the wind speed in m·s⁻¹ at the height of 10 m above the water surface, this paper uses the short-term wind speed data corresponding to the sampling moment. Sc number is the ratio of the dynamic viscosity of water to the diffusion rate of the gas molecules to be measured. Wanninkhof (1992) gives the relationship between the Sc number of N₂O gas and the water temperature:

$${\mathrm{Sc}}_{{\mathrm{N}}_2\mathrm{O}}=2055.6-137.11\times \mathrm{T}+4.3173\times {\mathrm{T}}^2-0.05435\times {\mathrm{T}}^3$$

(4)

where T is the water temperature.

2.4. Measurements of N₂O Emission and Dissolved N₂O

Both the emitted N₂O in the containers and the dissolved N₂O in the overlying water were measured. The emitted N₂O was determined by directly extracting gas samples from the headspace of each incubation experiment at 0 h, 12 h, 16 h, 20 h, 24 h, 36 h, 40 h, 44 h and 48 h [28]. The dissolved N₂O was determined by headspace equilibrium-gas chromatography [29]: briefly, the water sample was filled into 60 mL serum bottles, and 1 mL of 50% ZnCl₂ was added to inhibit the microbial activity [30]. 10 mL helium gas was injected into the serum bottles to act as a replacement for the water sample in order to create a headspace. The sample bottle was shaken vigorously for 30 min to equilibrate the gas-liquid phase in the bottle. After resting for 30 min to 1 h, the headspace volume was injected into the gas chromatograph for determination [31]. The concentration of N₂O was measured with a gas chromatograph (GC-2014C, Shimadzu, Japan) equipped with an electron capture detector (ECD).

2.5. Measurement of Denitrification Rate in Sediment

The denitrification rate of the sediment samples was measured at 0 h, 24 h and 48 h at the set temperatures using the slurry incubation and isotope pairing technique [32]. Fresh sediments were mixed with water in the ratio of 1:7 (sediment: water) and flushed with ultrahigh purity He for 30 min to promote the development of anaerobic sediment slurries. These slurries were pre-incubated in the dark at the set temperature for 36–48 h to remove background NO_x⁻ (NO₃⁻ and NO₂⁻) and dissolved oxygen (DO). After pre-incubation, the slurries were transferred to 12.5 mL tubes (Exetainers, Labco, High Wycombe, UK) via injectors. These tubes were divided into two groups: the first group was used to analyze F_n (¹⁵NO₃⁻/NO_x⁻), and the second was injected with a ¹⁵NO₃⁻ (99.6 atom%) solution to a final concentration of 100 μM. The tubes were incubated in the incubator (POMEX, Beijing, China) at the corresponding temperature, and microbial activity was stopped by adding 0.5 mL of 50% (v:v = 1:1) ZnCl₂ at 0 h and 2 h from the beginning of incubation. The ²⁹N₂ and ³⁰N₂ produced in the tubes were determined with a membrane inlet mass spectrometry (MIMS, HPR40, Hiden, UK), and the rates of denitrification were calculated as follows [33]:

$$\begin{gathered} {\mathrm{R}}_{\mathrm{D}}={\mathrm{D}}_{29}+2\times {\mathrm{P}}_{30} \\ {\mathrm{D}}_{29}={\mathrm{P}}_{30}\times 2\times \left(1-{\mathrm{F}}_{\mathrm{n}}\right)\times {\mathrm{F}}_{\mathrm{n}}{}^{-1} \end{gathered}$$

(5)

where R_D (nmol N g⁻¹ h⁻¹) represented the total rate of ¹⁵NO₃⁻—based denitrification, D₂₉ was the ²⁹N₂ production rate from denitrification, P₃₀ (nmol N g⁻¹ h⁻¹) was the total ³⁰N₂ production rate; F_n represented the fraction of ¹⁵N in total NO₃⁻.

The N₂O saturation was calculated based on the actual concentration of dissolved N₂O and the saturated concentration of N₂O at corresponding temperatures.

$$\sigma =\left(\mathrm{C}-{\mathrm{C}}_0\right)/{\mathrm{C}}_0$$

(6)

where C (nmol/L) represents the actual concentration of dissolved N₂O, C₀ is the saturated concentration of N₂O at gas-liquid equilibrium.

2.6. Statistical Analysis

To test significant differences between samples, one-way analysis of variance (ANOVA) was used for the normally distributed variables. The Pearson correlation or nonparametric Spearman correlation coefficients were then calculated to examine the relationship between samples. A significance level of p < 0.05 was used for all statistical analyses, which were carried out using the SPSS 22.0 software platform (SPSS Inc. Chicago, IL, USA).

Further explanation of DNA extraction, sequencing, and quantitative PCR (Table S1) can be found in the Supplementary Materials.

3. Results

3.1. N₂O Emission and Dissolved N₂O

During the 48 h incubation, higher levels of N₂O emission were observed at 35 °C (2.3 mmol N₂O/g soil average), which was higher than that at 25 °C (1.7 mmol N₂O/g soil average) and 15 °C (1.6 mmol N₂O/g soil average). The highest N₂O emission at 25 °C and 35 °C both occurred at 16 h, which were 2.3 and 3.5 mmol N₂O/g soil, respectively. At 15 °C, the highest N₂O emission was found at 36 h. After 36 h, the N₂O emissions were similar across all three temperatures, and all had a downward tendency (Figure 1a).

The dissolved N₂O showed a different pattern to the N₂O emission (Figure 1b); the average dissolved N₂O at 25 °C (140.9 nmol/L) was considerably higher than those at 35 °C (74.2 nmol/L) and 15 °C (70.6 nmol/L). The highest concentrations at 25 °C occurred between 12 h and 20 h, with concentrations around 215.9 and 250.3 nmol/L. The dissolved N₂O at 15 °C and 35 °C were low and similar to each other at an average of 70.6 and 74.2 nmol/L, respectively. Furthermore, the dissolved N₂O after 36 h was similar across the three temperatures.

The estimated N₂O exchange flux was greatest at 25 °C with an average flux of 35.1 nmol/m²·h, which was significantly higher than that at 15 °C (ANOVA, p = 0.085) and 35 °C (ANOVA, p = 0.006). Incubation at 35 °C demonstrated the lowest estimated N₂O exchange flux at 13.8 nmol/m²·h (Figure 1c, Table S2). The N₂O saturation was highest between 12 h and 20 h at 25 °C, ranging between 2820.3% and 3038.3% (Figure 1d). Although the dissolved concentrations were lower at 15 °C and 35 °C, they were nonetheless saturated at 785.2–847.5% and 1515.5–1626.6%, respectively.

3.2. Denitrification Rate in Sediment and the Concentration of Inorganic Nitrogen in Water

Higher mean denitrification rates in sediment were observed at 25 °C and 35 °C (12.5 and 12.8 nmol/g·h) than that at 15 °C (8.2 nmol/g·h). The denitrification rates increased significantly with incubation time, in which higher rates were observed at 24 h than at 0 h in all three temperatures (ANOVA, p = 0.031, 0.057 and 0.025, respectively). The denitrification rate at 48 h was also higher than that at 24 h at 25 °C. The anammox rates were lower than the denitrification rates and showed minor variation with the denitrification rates (Figure 2a–c).

The increase in the denitrification rate was in accordance with the decrease in NO₃⁻ concentration. Clear decreases in NO₃⁻ from 27.4 to 6.5 μmol/L and 27.4 to 0 μmol/L were observed at 25 °C and 35 °C, respectively (Figure 2d). NO₂⁻ was detected at a relatively low concentration in the overlying water at 35 °C with an increase from 2.4 to 3.9 μmol/L (Figure 2e). The NH₄⁺ content of the overlying water increased from 0 to 6.1 μmol/L at 15 °C, 0 to 12.8 μmol/L at 25 °C and 0 to 22.2 μmol/L at 35 °C before the 15 h, respectively. No clear trend was observed after 20 h of incubation (Figure 2f).

3.3. Abundance of N₂O-Related Functional Gene in Sediment

The abundance of functional genes related to denitrification (nirK, nirS, nosZ I and nosZ II) kept relatively stable and had no obvious trend over time (Figure S1). The abundance of the nirK gene varied from 3.43 × 10⁸ to 1.42 × 10⁹ copies/g dry soil, which was 1 order of magnitude higher than that of nirS. There was no significant difference in nirK gene abundance among the three temperatures. The abundance of the nirS gene was significantly higher (ANOVA, p = 0) at 15 °C (2.97 × 10⁸ copies/g dry soil) than at 25 °C (2.49 × 10⁸ copies/g dry soil) and 35 °C (2.41 × 10⁸ copies/g dry soil) (Figure 3a). The abundance of the nosZ II gene was 1 order of magnitude higher than nosZ I. The abundance of the nosZ II gene was significantly higher (ANOVA, p = 0.005) at 15 °C (1.55 × 10⁸ copies/g dry soil) than at 25 °C (1.34 × 10⁸copies/g dry soil) and 35 °C (1.33 × 10⁸ copies/g dry soil) (Figure 3b). The gene abundance nirS + nirK was 5 to 10 times higher than that of nosZ I + nosZ II, and there was no significant difference among the ratio of nirS + nirK/nosZ I + nosZ II at the three incubation temperatures (Figure 3c).

3.4. Factors Determining the N₂O Generation

At 15 °C, the dissolved N₂O and N₂O emissions were mainly related to the NO₃⁻ (Figure S2, Table S3). The dissolved N₂O in the overlying water increased from 66.4 nmol/L to 91.1 nmol/L, along with higher NO₃⁻ content (Figure 4a). At 25 °C, the dissolved N₂O, N₂O emission and the derived ratio parameters were mainly related to the ratio of nirS/nirK and nosZ I/nosZ II. Especially, the dissolved N₂O in the overlying water had a positive correlation with the ratio of nirS/nirK (p < 0.05) (Figure 4b). At 35 °C, the dissolved N₂O, N₂O emission and the derived ratios were mainly related to NO₃⁻, NO₂⁻, NO₃⁻/DIN and NO₂⁻/DIN. N₂O emission was positively correlated with the NO₃⁻ (p < 0.05) that the N₂O emission increased from 1.1 mmol N₂O/g soil to 3.5 mmol N₂O/g soil as the NO₃⁻ increased from 0 mg/L to 2 mg/L (Figure 4c).

4. Discussion

In this study, the average N₂O emission at 35 °C was 1.5 to 2.1 times higher than that at 25 °C and 15 °C, suggesting that the N₂O generation was temperature sensitive in freshwater sediment. This is in accordance with previous studies [34,35].

For example, the N₂O emissions were nearly 36 times higher in summer than in winter in polar freshwater lakes, which was ascribed to the enhanced rate of coupled nitrification-denitrification in summer [34]. Similarly, N₂O emissions were 2 times higher at 25 °C than that at 15 °C in soil, in which the faster growth of the microbial community induced a higher N₂O emission at higher temperatures [35]. Likewise, the N₂O emission was found to be more sensitive to temperature in wastewater treatment plants: the N₂O released from the water-gas interface was about three times higher at 35 °C than that at 25 °C, and the denitrification rate accordingly increased by 62% when the temperature increased from 25 °C to 35 °C [36]. Hence, higher temperatures directly increased the activity of denitrification as well as the N₂O emission [37,38]. Moreover, the increased temperatures could enhance N₂O emission by decreasing the organic carbon, thereby increasing the likelihood of incomplete denitrification and, therefore, also the likelihood for N₂O to be produced as an intermediate product [39].

It was noted that the highest saturation and estimated exchange flux of N₂O were observed at 25 °C, which were both about 2 times higher than those at 35 °C and 15 °C. The dissolved N₂O was in a state of oversaturation (667.3%, 1811.2% and 1408.4% at 15 °C, 25 °C and 35 °C, respectively) in all samples, suggesting that N₂O had a high potential for being released into the atmosphere. The saturation of N₂O in the current study was higher than those reported from natural habitats, including freshwater reservoirs, rivers and estuaries, with a saturation of 84% to 745%, 152–451% and 45–2187%, respectively [40,41]. This could be due to the relatively stable and inert environmental conditions in the incubation, allowing for the accumulation of N₂O in water. The microbial activity and the solubility of N₂O in the water were two key factors determining the dissolved N₂O in in situ conditions. Higher temperatures stimulated microbial activity and generated more N₂O [35] but decreased the solubility of N₂O in water [42]. Henry’s constant, which is also called the air-water partition coefficient, rested on the temperature condition [43]. Henry’s constant of N₂O in water increased from 4146 k_H/Pa·m³·mol⁻¹ to 6010 k_H/Pa·m³·mol⁻¹ when the temperature increased from 25 °C to 40 °C [43]. Theoretically, the N₂O solubility in pure water decreased by 23% when the temperature increased from 25 °C to 35 °C [44]. In a field survey, the N₂O solubility was 125–385% less than in the current ex-situ study, which can be attributed to more N₂O being diffused into the headspace and the slower re-dissolution of N₂O caused by higher accumulation in the gas phase at higher temperature [36]. However, it is important to note the dissolved N₂O merely suggests a potential for emission and not an actual emission per se. The N₂O in water still had a great probability of being reduced by microorganisms carrying the nosZ gene before being emitted into the atmosphere [21].

The increase in temperature might influence many other factors such as soil organic carbon, nutrient availability and mineralization rate, etc. For example, the temperature sensitivity of soil organic carbon is lower in subtropical forests but higher in temperate forests. The C:N ratio of soil is significantly and positively correlated with organic carbon temperature sensitivity [45].

In this study, there was a significant increase in denitrification rates over time, and a positive correlation was observed between the denitrification rate and N₂O emission at the three temperatures, which indicated that the denitrification processes might dominate the N₂O emission. This is in accordance with previous studies in freshwater [46], tidal wetlands [47], riparian zones [48] and urban rivers [49], where the higher denitrification rates corresponded to higher N₂O fluxes. In addition, the habitats in which denitrification dominates the N₂O emission are usually sinks for NO₃⁻ [48]. It has previously been observed that the NO₃⁻ content shows a strong correlation with the N₂O emission in many habitats, including deep wells [50], freshwater rivers and lakes, etc. [51,52], and indeed, the NO₃⁻ content has been used as an indicator for the N₂O emission [53]. In this study, the NO₃⁻ showed a sharp decline, especially at high temperatures, presenting a negative relationship with the N₂O emission, which confirmed the dominant role of denitrification in N₂O emission. However, it cannot be ruled out that the DNRA pathway does not play a role since an increase of NH₄⁺ was concurrently observed. In the present study, the total nir/nos ratio was between 5 and 10, indicating that the microbial community had a higher potential to produce N₂O than to reduce it [52,54]. It was confirmed by the high ratio of nirK to nirS, which was 1.2 to 2.4, that nirK-type denitrifies are more likely to perform incomplete denitrification and thereby contribute more to N₂O emissions [21].

5. Conclusions

The present study showed that the highest N₂O emission in freshwater sediment is observed at an elevated temperature of 35 °C. This was demonstrated through a series of incubation experiments with a temperature gradient at 15 °C, 25 °C and 35 °C. In contrast, the dissolved N₂O in the water column had a different pattern than that of N₂O emission; the highest concentration was namely observed at 25 °C, indicating that the highest potential of N₂O emission occurs at moderate temperatures. The denitrification rates significantly increased during incubation, while the rates at 25 °C and 35 °C were much greater than that at 15 °C, which coincides with the N₂O emissions at the three temperatures. The NO₃⁻ content was a key indicator of denitrification, which decreased along with the increase in N₂O emissions, thereby presenting a negative relationship between them. The nirK-type denitrifier dominated denitrification and N₂O generation, while the nosZ II-type denitrifier dominated N₂O reduction. The current analysis indicates that high temperatures (35 °C) may enhance denitrification-derived N₂O emissions, and moderated temperatures (25 °C) have higher dissolved N₂O, making it a potential source of N₂O emissions from freshwater ecosystems.