Influence of Acetylene Concentration on N₂O and N₂ Emissions from an Intensive Vegetable Soil under Anoxic and Oxic Conditions

Abstract

Acetylene (C₂H₂) is often employed to assess soil total denitrification (N₂O + N₂) due to its ease of implementation. However, this technique underestimates soil denitrification in soils with low nutrient contents, particularly those supporting grain yields. To our knowledge, there are limited studies that have specifically investigated the impact of C₂H₂ on nutrient-rich vegetable soils, especially concerning the emissions of N₂ and N₂O and the nitrogenous gas product ratio (i.e., N₂O/(N₂O + N₂)). In this study, we conducted both anoxic and oxic incubations at various C₂H₂ concentrations (0%, 0.01%, and 10%, v/v) and utilized a robotized sampling and analysis system to quantify soil N₂, N₂O, and CO₂ emissions. Our findings revealed that the cumulative N₂O production in soil treated with 10%C₂H₂ was significantly lower than that in soil treated with 0.01%C₂H₂ and soil without C₂H₂. Contrarily, high concentrations of C₂H₂ (10%, v/v) led to increased N₂ production. Similar trends were observed under oxic conditions, where 10%C₂H₂ concentration did not enhance N₂O production but markedly increased N₂ and CO₂ emissions. Moreover, the N₂O/(N₂O + N₂) product ratio was notably higher in soils treated with 0%C₂H₂ compared to the 10%C₂H₂ treatment under anoxic conditions. These findings indicate that high concentrations of acetylene could facilitate the reduction of N₂O to N₂ and lead to underestimated soil total denitrification in vegetable soil, regardless of anoxic or oxic conditions. This discovery underscores the drawbacks when employing high concentrations of acetylene to evaluate actual total denitrification in intensive greenhouse vegetable soils, highlighting the necessity for further investigation into alternative methodologies.

1. Introduction

Nitrogen (N) is an essential nutrient that plays a crucial role in regulating ecosystem functioning and ensuring food production and security. Increased food and energy production has led to a significant increase in N fertilizer usage, including synthetic fertilizer and manure [1], causing accelerated N cycling in agricultural soils. Different agroecosystems employ various fertilization regimes, mostly depending on crop types. In intensive greenhouse vegetable production systems, the application of N fertilizer exceeds 2000 kg N ha⁻¹ yr⁻¹ in two cropping seasons, far beyond plant needs [2,3]. This excessive practice results in high nitrate leaching [4] and gaseous N losses from soils, partially in the form of nitrous oxide (N₂O), a greenhouse gas [5], and denitrified dinitrogen (N₂) [6,7]. These N losses are crucial in determining N availability for plants and microbes [8,9]. Measuring gaseous N losses from soils is challenging due to the high background concentration of atmospheric N₂ [10,11].

Various approaches have been developed to quantify N₂ losses from soils, such as (a) the acetylene (C₂H₂) inhibition technique [12], (b) ¹⁵N traces [13], (c) the N₂/Ar technique [14], and (d) helium/O₂ atmosphere exchange [15]. The weaknesses and strengths of these methods have been reviewed [10,16]. Since its early development, the acetylene inhibition technique has become the most widely used indirect method to determine soil denitrification due to its simplicity, short incubation time, low cost, and good reproducibility [16,17]. For example, nearly half of 236 studies used the acetylene inhibition technique to measure N₂ production from the complete denitrification process [16]. Moreover, different concentrations of acetylene (C₂H₂) can be utilized to differentiate between nitrification and denitrification as sources of N₂O in soil [18,19,20].

Typically, low concentrations of C₂H₂ (0.01%, volume ratio, 10 Pa) are employed to evaluate the contribution of autotrophic nitrification by inhibiting soil ammonia monooxygenase (AMO) activity [21,22]. High concentrations of C₂H₂ (10%) can indirectly measure soil denitrification rates and N₂ emissions by inhibiting ammonia oxidation while preventing the reduction of N₂O to dinitrogen (N₂) via denitrification [23,24]. Nonetheless, the C₂H₂ inhibition technique also has certain limitations, leading to an underestimation of soil denitrification (i.e., N₂O + N₂ production) [25,26] and N₂ emissions [27]. This underestimation may be attributed to diffusion restriction of C₂H₂ to microsites, potential effects of microbial decomposition, and incomplete inhibition of N₂O reductase. These biases have mostly been observed in experiments conducted in soils with low soil nutrient and organic matter content, such as grain yield soils [26,28]. However, it remains unclear whether the C₂H₂ inhibition technique also underestimates soil denitrification in greenhouse vegetable soils with high soil nutrient levels (e.g., nitrate). Furthermore, the effects of the C₂H₂ inhibitor on soil N₂ emissions in greenhouse vegetable soils are not well understood.

The primary objective of this study is to evaluate the effects of C₂H₂ on soil denitrification and N₂ production in a greenhouse vegetable cropping system. To attain this, soil samples were collected from a long-term fertilization site, established in 2004, located in the Shouguang area. We conducted laboratory incubation experiments under both anoxic and oxic conditions with different acetylene concentrations. The concentrations of N₂O and N₂ were quantified utilizing a robotic sampling and analysis system, ensuring accuracy and precision in our measurements.

2. Materials and Methods

2.1. Sampling Site

Soil samples were collected from a site in Luozhuang Village, Gucheng Street, Shouguang City, Shandong Province (36°55′ N, 118°45′ E) that was established by China Agricultural University in 2004. The greenhouse vegetable soil there is characterized by a sandy loam texture. The study area exhibits a mean annual air temperature of 12.4 °C and mean annual precipitation of 558 mm. More details on the layout of the experiment are provided in the previous literature [29]. In brief, there were two growing seasons per year with continuous cropping of tomato: the autumn–winter (AW) and winter–spring (WS) seasons. The AW season began in early August and ended the following January, while the WS season started in early February and ended in the middle of June. A brief summer fallow period lasted less than two months. The tomato vines were removed from the greenhouse at the final harvest according to conventional practice. Conventional fertilization treatment with three replicates was selected in the vegetable greenhouse. Prior to tomato planting, chicken manure was utilized as a basal fertilizer at rates of 270 and 190 kg N ha⁻¹ in the AW and WS seasons, respectively. The annual application rate of chemical nitrogen fertilizer was 1200 kg N ha⁻¹. Phosphorus and potassium fertilizers were applied at 150 kg P₂O₅ ha⁻¹ and 520 kg K₂O ha⁻¹, respectively, in each growing season. Furrow irrigation and conventional fertilization followed, in which the fertilizers were dissolved and applied with the furrow irrigation water. Irrigation time and amount of irrigation were determined by farmers based on local weather, soil moisture contents, and crop growth. In each growing season, 10 to 11 irrigation events took place, and 540 mm to 560 mm water was supplied.

2.2. Anoxic and Oxic Incubations and Gas Monitoring

To investigate the effects of varying acetylene concentrations on N₂O, N₂, and CO₂ emissions, soil samples (0–20 cm) were collected at the end of the AW season from conventional fertilization treatment. Fresh soil samples were sieved through a 2 mm mesh to remove roots and other debris and were thoroughly mixed before being stored at 4 °C for use. The laboratory incubation comprised six treatments, considering the oxygen levels (anoxic and oxic conditions) and acetylene concentrations (0%, 0.01%, and 10%, v/v). For anoxic incubations, there were three groups: a control without inhibitor (0%C₂H₂, anoxic environment with 99.999% helium gas), a low acetylene concentration (0.01%C₂H₂, anoxic environment with 0.01% C₂H₂ (v/v) in the headspace), and a high acetylene concentration (10%C₂H₂, anoxic environment with 10% C₂H₂ in the headspace). Under oxic conditions, the design included a control without inhibitor (0%C₂H₂, oxic environment with 18% O₂ in the headspace), a combination of a low concentration of C₂H₂ and O₂ (0.01%C₂H₂, oxic environment with 18% O₂ and 0.01% C₂H₂ in the headspace), and a combination of a high concentration of C₂H₂ and O₂ (10%C₂H₂, oxic environment with 18% O₂ and 10% C₂H₂ in the headspace). Each treatment was replicated 5 times to minimize errors. Soil N₂O, N₂, and CO₂ emissions were measured with a robotic incubation system at a constant temperature of 20 °C. All soils were analyzed for ammonium, nitrite, and nitrate after incubation.

The robotic incubation system consisted of an automatic sampling module and a gas analysis module (Agilent 7890A gas chromatograph, Santa Clara, CA, USA). The automatic sampling module consisted of a headspace autosampler (CTC GC-Pal) and a bidirectional helical peristaltic pump (Gilson Model 222, Gilson, Corbonod, France). The gas analysis module included an electron capture detector (ECD), a thermal conductivity detector (TCD), and a flame ionization detector (FID) the gas chromatograph, which could monitor changes in N₂O (ECD, TCD), N₂ (TCD), and CO₂ (TCD) gas concentrations. Specifically, ECD data were used for N₂O concentrations below 10 ppmv, and TCD data were used for N₂O concentrations above 10 ppmv. Further details on the robotic incubation system, such as the periodic sampling and analysis of the headspace gas, are described by Molstad et al. [30]. The specific experimental procedure was as follows: Approximately 12.0 g of dried soil was weighed and added to 120 mL serum flasks. A certain amount of deionized water was added to the serum flasks using a syringe to adjust the soil moisture content to 20%. The serum flasks were sealed with rubber stoppers and aluminum caps. A vacuum-inflation system (Beijing Ferren Science & Technology Co., Ltd., Beijing, China) was used to flush the serum flasks with high-purity helium (99.999%); a mixture of 0.01% C₂H₂ and helium; a mixture of 10% C₂H₂ and helium; a mixture of 18% O₂ and helium; a mixture of 18% O₂, 0.01% C₂H₂, and helium; and a mixture of 18% O₂, 10% C₂H₂, and helium. Each serum flask was flushed five times and then filled with the corresponding gas and kept under overpressure for 3 min. The pressure in the headspace of the serum flasks was equilibrated with atmospheric pressure using a syringe filled with deionized water. All the flushed serum flasks were placed in a robotized incubation/monitoring system to conduct a 40 h incubation period. During this period, the concentrations of N₂O, N₂, and CO₂ were monitored at intervals of 8 h. The production rates of these gasses were calculated based on their concentrations, as recorded by the robotic incubation system, compared with known standard gas concentrations. The AIT-bias was calculated from the following equation:

$$\mathrm{AIT}\text{-}\mathrm{b}\mathrm{ias}={\displaystyle \frac{{(\mathrm{N}}_2\mathrm{O}{)}_{+{\mathrm{C}}_2{\mathrm{H}}_2}-{(\mathrm{N}}_2\mathrm{O}+{\mathrm{N}}_2{)}_{-{\mathrm{C}}_2{\mathrm{H}}_2}}{{(\mathrm{N}}_2\mathrm{O}+{\mathrm{N}}_2{)}_{-{\mathrm{C}}_2{\mathrm{H}}_2}}}\times 100\%$$

where the parameters (N₂O)_+C2H2 and (N₂O + N₂)_{− C2H2} represent the N₂O production rate (in nmol g⁻¹ h⁻¹) and the total N₂O and N₂ production rate (in nmol g⁻¹ h⁻¹) in the presence and absence of acetylene, respectively. The N₂O product ratio (N₂O/(N₂O + N₂)) was calculated as the ratio of the N₂O production rate to the total N₂O and N₂ production rate [28].

2.3. Analysis of Soil Physical and Chemical Properties

Soil total nitrogen (TN) was determined using a Kjeldahl nitrogen analyzer. Soil pH was measured potentiometrically in deionized water (1:2.5 w/v) using a combination pH electrode (PHS-3E, Shanghai Precision & Scientific Instrument Co., Ltd., Shanghai, China). Soil organic carbon (SOC) was determined using the potassium dichromate–sulfuric acid oxidation titration method. Available phosphorus was determined using the molybdenum–antimony anti-absorption spectrophotometry method after extraction with 0.5 mol L⁻¹ NaHCO₃. Soil moisture was determined by drying the soil in an oven at 105 °C for 24 h. Soil ammonium (NH₄⁺) and nitrate (NO₃⁻) were determined by extraction with 1 mol L⁻¹ KCl followed by measurement using a continuous flow analyzer (TRAACS 2000, Bran Luebbe, Norderstedt, Germany). Soil NO₂⁻ was measured using the sulfanilamide method [31]. In this study, all the reported calculations were conducted based on dry soil mass. The major properties of the greenhouse vegetable soil used are summarized in

Table 1. Basic soil characteristics prior to incubation.

pH	Soil Organic Carbon (g kg−1)	Total Nitrogen (g kg−1)	Olsen-P (mg kg−1)	Exchangeable Potassium (mg kg−1)	NH4+-N (mg kg−1)	NO2−-N (mg kg−1)	NO3−-N (mg kg−1)
6.54 ± 0.02	9.56 ± 0.38	1.55 ± 0.036	232 ± 0.75	240 ± 2.7	21.2 ± 0.24	0.066 ± 0.002	297 ± 44

.

2.4. Statistical Analysis

SPSS software (version 20.0) was used to perform one-way analysis of variance (ANOVA) with least significant difference (LSD). The experimental data were plotted using Sigmaplot 12.5; dashed lines represent mean values, solid lines represent medians, and bar graphs represent mean values ± standard deviations.

3. Results

3.1. Cumulative N₂O Production

As depicted in Figure 1, a markedly higher cumulative N₂O production was observed during anoxic incubation than during oxic incubation, indicating that the anoxic conditions greatly enhanced N₂O emissions. The cumulative N₂O production under anoxic conditions was 110.0, 107.6, and 0.37 nmol N g⁻¹ in the 0%C₂H₂, 0.01%C₂H₂, and 10%C₂H₂ treatments, respectively. Notably, a high concentration of acetylene (i.e., 10%C₂H₂) did not result in increased N₂O production compared to the 0.01%C₂H₂ treatment. Under oxic conditions, the cumulative N₂O production was 0.046, 0.16, and 0.044 nmol N g⁻¹ for the 0%C₂H₂, 0.01%C₂H₂, and 10%C₂H₂ treatments, respectively. The cumulative N₂O production in the 0.01%C₂H₂ treatment was significantly higher than those in the other treatments (p < 0.05). Notably, similar to anoxic incubation, a high acetylene concentration did not elevate N₂O production during oxic incubation.

3.2. Cumulative N₂ Production

As illustrated in Figure 2, the cumulative N₂ production was 44.0, 88.2, and 297.2 nmol N g⁻¹ for the 0%C₂H₂, 0.01%C₂H₂, and 10%C₂H₂ treatments, respectively. The addition of acetylene concentrations of 0.01% and 10% significantly increased N₂ production compared to the 0%C₂H₂ treatment (p < 0.05), indicating that acetylene stimulated nitrate reduction. Moreover, under oxic conditions, the 10%C₂H₂ treatment resulted in significantly higher cumulative N₂ production than the 0%C₂H₂ and 0.01%C₂H₂ treatments (p < 0.05), which further supports the notion that high concentrations of acetylene can promote N₂ production.

3.3. Nitrogenous Gas Production

The production of nitrogenous gasses, namely N₂O and N₂, was calculated as the sum of the production of the individual gas at the end of incubation (Figure 3). Under anoxic conditions, the treatment with a 10%C₂H₂ concentration exhibited significantly greater gas production compared to the treatments without C₂H₂ and with 0.01%C₂H₂ (p < 0.05). These findings suggest that a high C₂H₂ concentration markedly enhances nitrogenous gas production under anoxic conditions. Likewise, during oxic incubation, the 10%C₂H₂ treatment demonstrated significantly higher nitrogenous gas production compared to the other treatments (p < 0.05).

3.4. N₂O and N₂ Production Rates and N₂O/(N₂O + N₂) Product Ratio

As shown in

Table 2. N₂O and N₂ production rates, N₂O/(N₂O + N₂), and bias in greenhouse vegetable soil under anoxic and oxic conditions with varying acetylene levels.

Production Rate	Anoxic Conditions			Oxic Conditions
	0%C2H2	0.01%C2H2	10%C2H2	0%C2H2	0.01%C2H2	10%C2H2
N2O-N (nmol g−1 h−1)	2.75 ± 0.10 a	2.69 ± 0.17 a	0.01 ± 0.005 b	0.0011 ± 0.0003 b	0.004 ± 0.0029 a	0.0011 ± 0.0001 b
N2-N (nmol g−1 h−1)	1.10 ± 0.11 b	2.21 ± 1.49 b	7.44 ± 2.07 a	1.32 ± 0.25 b	1.56 ± 0.16 b	5.91 ± 1.61 a
(N2O + N2)-N (nmol g−1 h−1)	3.85 ± 0.20 b	4.90 ± 1.47 b	7.44 ± 2.07 a	1.32 ± 0.25 b	1.56 ± 0.16 b	5.91 ± 1.61 a
N2O/(N2O + N2) (%)	71.47 ± 1.66 a	58.25 ± 14.6 b	0.13 ± 0.08 c	0.09 ± 0.03 b	0.26 ± 0.21 a	0.02 ± 0.005 b
AIT-bias (%)		−30.1 ± 4.50 b	−99.8 ± 0.12 a		−99.7 ± 0.22 a	−99.9 ± 0.01 a

Note: Different letters (e.g., ‘a’, ‘b’, ‘c’) represent significant differences (p < 0.05) at various acetylene levels under the same oxygen condition.

, under anoxic conditions, the treatment with a high concentration of C₂H₂ (i.e., 10%C₂H₂) exhibited a significantly lower N₂O production rate than that with 0.01%C₂H₂ and that without C₂H₂. Clearly, the high concentration of C₂H₂ corresponded to a significant increase in the N₂ production rate (p < 0.05), consequently lowering the N₂O/(N₂O + N₂) product ratio and increasing the AIT-bias. This finding suggests a substantial underestimation of nitrogen gaseous emissions from the soil. Under oxic conditions, a similar effect was observed in that the high concentration of acetylene (10%, v/v) also led to underestimated nitrogen gaseous loss (N₂O + N₂) in greenhouse vegetable soil.

3.5. Cumulative CO₂ Production

As shown in Figure 4, under anoxic conditions, the results of cumulative CO₂ production were 337.3, 353.3, and 403.2 nmol C g⁻¹ in the 0%C₂H₂, 0.01%C₂H₂, and 10%C₂H₂ treatments, respectively. When compared with the 0%C₂H₂ and 0.01%C₂H₂ treatments, a high C₂H₂ concentration (i.e., 10%C₂H₂) significantly increased CO₂ production (p < 0.05). A similar trend was observed under oxic conditions, with a high concentration of C₂H₂ (10%) treatment exhibiting the highest cumulative CO₂ production (445.5 nmol C g⁻¹) compared to the other treatments.

3.6. Nitrite and Ammonium Concentration

Under anoxic conditions, the nitrite (NO₂⁻) accumulated concentration was 2.36, 2.18, and 0.095 mg N kg⁻¹ in the 0%C₂H₂, 0.01%C₂H₂, and 10%C₂H₂ treatments, respectively (Figure 5a). The accumulations of NO₂⁻ in the 0%C₂H₂ and 0.01%C₂H₂ treatments were significantly (p < 0.05) higher than that in the 10%C₂H₂ treatment, showing that higher acetylene concentration could slow down NO₂⁻ accumulation, possibly by enhancing NO₂⁻ reduction during anoxic incubation. Conversely, NO₂⁻ concentration was significantly higher in the treatment with a high acetylene concentration (i.e., 10%C₂H₂) than in that without acetylene (i.e., 0%C₂H₂) during oxic incubation (Figure 5b, p < 0.05).

As shown in Figure 5c, ammonium (NH₄⁺) accumulation during anoxic incubation ranged from 23.1 to 29.7 mg N kg⁻¹. These NH₄⁺ accumulations were increased at the end of incubation compared with the initial NH₄⁺ concentration (21.2 mg N kg⁻¹, Table 1). Under oxic conditions, no significantly higher NH₄⁺ accumulations were observed among the 0%C₂H₂, 0.01%C₂H₂, and 10%C₂H₂ treatments (Figure 5d). Additionally, there were no significant differences in nitrate (NO₃⁻) accumulations among all treatments, regardless of anaerobic or aerobic conditions, indicating that acetylene did not markedly affect soil nitrate concentration at the end of incubation.

4. Discussion

4.1. Effect of Acetylene Inhibitor on Soil N₂O and N₂ Production

Under anoxic conditions, our results showed that a high concentration of C₂H₂ (i.e., 10%) had a significant impact on reducing the cumulative N₂O production in a greenhouse vegetable soil with high nitrate concentration compared with a 0%C₂H₂ treatment. This finding diverges from earlier studies [28,32], which reported an increase in N₂O concentration with C₂H₂ treatment over time. Smith et al. [28] noted that the effectiveness of C₂H₂ in inhibiting the reduction of N₂O to N₂ is enhanced at high NO₃⁻ concentrations. The prevailing view is that 10% (v/v) C₂H₂ leads to high N₂O concentrations by inhibiting the N₂O reductase enzyme in denitrifying microbes, as established by Knowles [27] and by N₂ production assessed by measuring the difference in N₂O produced between C₂H₂-treated and untreated flasks [33,34]. However, our data suggest that high C₂H₂ concentrations actually promote the reduction of N₂O to N₂ under anoxic conditions (Figure 2), challenging the findings of Qin et al. [28], which indicated lower N₂ increases with C₂H₂ treatment. There are several reasons for the increased N₂ production at high C₂H₂ levels. First, some denitrifiers are not sensitive to C₂H₂, weakening its inhibitory effect [25,35]. Second, some products (e.g., acetate, ethanol) of the degradation of acetylene via soil microorganisms can be utilized by denitrifying bacteria [36,37], promoting the reduction of N₂O. And the availability of carbon is likely to be a major limiting factor in greenhouse vegetable soil during denitrification [6]. In oxic conditions, elevated C₂H₂ levels did not escalate N₂O production but significantly boosted N₂ yields, as indicated in Figure 2. This could be ascribed to the micro-anaerobic conditions induced by C₂H₂ degradation among soil microorganisms [38], which in turn may have enhanced the reduction of N₂O to N₂.

4.2. Effect of Acetylene Inhibitor on Actual Soil Denitrification and Gaseous Product Ratio

Our findings demonstrated that a high concentration of C₂H₂ significantly influenced the production of nitrogenous gas (i.e., the sum of N₂O and N₂ production) in greenhouse vegetable soils, irrespective of anoxic or oxic conditions. Specifically, the presence of high C₂H₂ (10%) led to enhanced actual soil denitrification (Figure 3). This raises questions about the efficacy of using high C₂H₂ concentrations as an inhibitor method to evaluate actual soil denitrification potential. Our data showed that relying on high C₂H₂ concentrations may unpredictably underestimate actual soil denitrification, as C₂H₂ promotes the reduction of N₂O to N₂. This is corroborated by previous studies that have noted incomplete inhibition of N₂O reduction to N₂ when employing the C₂H₂ inhibition technique as opposed to the ¹⁵N-nitrate tracer or alternative approaches [24,26,28,38,39]. In our study, under anoxic conditions, there was a significant reduction in the N₂O/(N₂O + N₂) product ratio in greenhouse vegetable soil due to the presence of high C₂H₂ concentrations, as detailed in Table 2. This result aligns with previous findings from grain field soils, which also noted a decrease in the N₂O/(N₂O + N₂) product ratio in response to C₂H₂ [28]. This alignment might be due to high concentrations of NO₃⁻ inhibiting the reduction of N₂O to N₂ during denitrification, as NO₃⁻ is favored over N₂O as an electron acceptor [40,41,42,43]. The accumulation of NO₃⁻ in greenhouse vegetable soil, as shown in Table 1, may thus have impeded N₂O consumption, resulting in a higher N₂O/(N₂O + N₂) product ratio in the absence of C₂H₂ treatment. On the other hand, denitrifying microorganisms can metabolize certain byproducts of acetylene degradation [27], potentially accelerating the reduction of NO₃⁻ and N₂O in the soil [44]. This mechanism could contribute to a significant underestimation of total denitrification in vegetable soil, as evidenced by the data presented in Table 2.

4.3. Effect of Acetylene Inhibitor on Soil Inorganic Nitrogen

Denitrification processes are characterized by a sequential reduction pathway, typically proceeding from NO₃⁻ to NO₂⁻, then to nitric oxide (NO), N₂O, and finally to N₂, with NO₃⁻ reduction occurring first under completely anoxic conditions [43]. Under scenarios where organic carbon is scarce, competition for electrons among the key denitrification reductases (e.g., Nar, Nir, Nor, and Nos) becomes intense. Our study showed that the concentration of nitrite (NO₂⁻) was considerably lower in the treatment with 10%C₂H₂ compared with the treatment without C₂H₂ under anoxic conditions (Figure 5). This difference can be attributed to certain byproducts of acetylene degradation serving as an alternative carbon source that enhanced the reduction of NO₂⁻. Conversely, under oxic conditions, an elevated concentration of NO₂⁻ was detected in the 10%C₂H₂ treatment, which can be primarily ascribed to the increased anoxic microsites resulting from C₂H₂ degradation promoting denitrification and nitrate reduction. Additionally, the accumulation of ammonium in the greenhouse vegetable soil at the conclusion of anoxic incubation suggests that dissimilatory nitrate reduction to ammonium may also have occurred, a process supported by findings from Blackmer and Bremner [40] and further studies [45,46]. Furthermore, no significant differences in nitrate production were observed between the treatments with and without C₂H₂, which could be attributed to the substantial background concentration of NO₃⁻ in the soil. Lastly, the short incubation period resulted in minimal nitrogenous gas production, having a negligible effect on the overall NO₃⁻ dynamics.

5. Conclusions

This study revealed that an elevated concentration of C₂H₂ (10%, v/v) substantially boosted the generation of N₂ and the overall yield of nitrogenous gasses (i.e., N₂O + N₂) in greenhouse vegetable soils under anoxic conditions. Consequently, there was a significant reduction in the N₂O/(N₂O + N₂) product ratio. Under oxic conditions, a similar trend was observed; high C₂H₂ concentrations did not elevate N₂O production but markedly increased N₂ production. These results suggest that high levels of acetylene can expedite the conversion of N₂O to N₂, potentially leading to an underestimation of the actual total denitrification occurring in greenhouse vegetable soil, irrespective of anoxic or oxic conditions. Given these findings, it is imperative to carefully reconsider the use of C₂H₂ as an inhibitor when assessing the denitrification potential of soils within this specific cropping system. The capacity of high C₂H₂ concentrations to promote the reduction of N₂O to N₂ is a significant factor that merits further investigation and contemplation.