Arrhenius equation construction and nitrate source identification of denitrification at the Lake Taihu sediment − water interface with 15 N isotope

Total nitrogen in Taihu Lake, China has gradually decreased since 2015 while the total phosphorus concentration has exhibited an increasing trend, indicating an asynchronous change. The dominant nitrogen removal process in freshwater ecosystems is denitrification which primarily occurs at the sediment–water interface. In this study, 15 N isotope incubation experiments were attempted to analyze the effect of water temperature on denitrification, to construct the regional denitrification Arrhenius equations considering water temperature, and to identify the nitrate source of denitrification in Lake Taihu sediments. The results indicated that the potential N2 production rates and denitrification rates generally decreased in the west to east direction, which was significantly positively correlated with the nitrate concentration of overlying water by Pearson correlation coefficient analysis (P < 0.05). In addition, when the water temperature was lower than 30 °C, the rates of the potential N2 production and denitrification were higher with an increase in water temperature, but when the water temperature was overhigh, denitrification was inhibited. The ratio of the total denitrification rate of nitrate from the water column in the sediment to the total denitrification rate during the incubation experiment was above 0.5 at each sampling site. This indicated that the denitrification in the Lake Taihu sediment primarily occurred at the expense of nitrate from the water column. The research results of Arrhenius equation construction and nitrate source identification of denitization can be applied to improve the accuracy of water quality model of Taihu Lake, which is of great significance to improve Taihu Lake water quality, and can act as a reference for the water environment treatment of other shallow eutrophic lakes in China and abroad.


Introduction
Nitrogen enters a lake as inorganic and organic nitrogen through atmospheric deposition, surface runoff, and biological nitrogen fixation, and is absorbed and assimilated by algae, aquatic plants, and benthos (Cottingham et al. 2015;He et al. 2020). These nutrients can be released into the water column through leakage or mortality by organisms (Zhang et al. 2009a, b;Jiang et al. 2019). Human activities have accelerated the input of reactive nitrogen to the biosphere (Frostegard et al. 2021). The increasing input of nitrogen into water is one of the principal anthropogenic stressors leading to lake water eutrophication (Chen et al. 2012;Li et al. 2020).
Denitrification, anaerobic ammonium oxidation, and dissimilatory nitrate reduction to ammonium are crucial pathways of dissimilatory nitrate reduction in aquatic ecosystems (Kuypers et al. 2003;Jiang et al. 2019Jiang et al. , 2020Ahmad et al. 2021). Therefore, studies on the effect of denitrification at the sediment − water interface are vital for understanding lake water eutrophication. Denitrification refers to the process in which microorganisms reduce nitrate and nitrite to gaseous nitrogen compounds and nitrogen under anoxic conditions. It is the primary nitrogen removal process in freshwater ecosystems, occurring at the sediment − water interface (Saunders and Kalff 2001;Veraart et al. 2011), and is greatly affected by water temperature. Liao et al. (2018) pointed that temperature variation resulted in the shift of microbial community structure and diversity, and exerted effects on the abundance of denitrification function genes, nirK, nirS, arG, and nosZ. Wang et al. (2018) found that the low-temperature shocks down-regulated the expression of denitrifying genes, and nitrate and nitrite oxide reduction rates generally followed the order: 34 °C > 25 °C > 20 °C > 12 °C > 4 °C. Additionally, water temperature can also directly or indirectly affect denitrification by affecting the dissolved oxygen concentration, nutrient release rate, etc. (Gebremariam et al. 2021;Minuti et al. 2021;Wang et al. 2021).
At present, the determination of denitrification rate mainly includes acetylene inhibition method, nitrogen direct quantification method, and isotope method. However, Wu et al. (2019) considered that acetylene inhibition technique was unsuitable for quantifying the denitrification rates, mainly because of acetylene and oxygen would catalyze the decomposition into nitric oxide when they existed together, thus affecting the determination results. The nitrogen direct quantification method is used to directly measure the nitrogen produced by denitrification, which requires high airtightness of the experimental device, but it is difficult to ensure the airtightness of the system in actual experiments. The nitrogen isotopes method is a highly sensitive method that uses nitrogen isotopes as tracers and has been widely used for understanding inorganic nitrogen sources, migration, and transformation in various environments (Bu et al. 2017;Jin et al. 2017;Meng et al. 2021). And Granger and Wankel (2016) proposed that the natural abundance of nitrogen (δ 15 N) and oxygen isotopes of nitrate (δ 18 O) were important tools for evaluating the sources and transformations of natural and contaminant nitrate in the environment. Lewicka-Szczebak et al. (2014) quantified denitrification in arable soils based on stable isotope analyses of emitted N 2 O (δ 15 N and δ 18 O). Kim et al. (2018) analyzed δ 15 N and δ 18 O using a Finnigan MAT delta plus XL isotope ratio mass spectrometer at the Isotope Science Laboratory to assess denitrification in a hyporheic zone. In addition, the nitrogen isotope method can not only accurately measure the denitrification rate, but also calculate the coupled denitrification rate and the uncoupled denitrification rate, which can be used to identify the nitrate source of denitrification (Steingruber et al. 2001).
In order to better understand the complex water environment problems, many scholars have built water environment mathematical models to quantitatively study the changes of chemical and ecological factors in the water environment. Cheng et al (2020) reported that the denitrification rate in the Environmental Fluid Dynamic Code (EFDC) model was a sensitive parameter of nitrogen cycle in water. However, in application, the denitrification rate parameters of the model mostly adopted the inherent recommended values, without considering the actual situation of the lake. Besides, in the EFDC control equation, temperature, and denitrification rate are described as consistent with Arrhenius equation (Ji 2017).
Therefore, this paper expected to obtain the denitrification Arrhenius equation considering temperature and main source of denitrification nitrate of shallow eutrophic lake Taihu through isotope culture experiment, and can provide some scientific basis for building a refined mathematical model of Taihu Lake water environment.

Study area description and sample collection
Lake Taihu, located in the eastern plain of China (Jiang et al. 2020), is a large eutrophic shallow (area 2338 km 2 , mean depth 1.9 m) freshwater lake (Zhang et al. 2009a, b). Lake Taihu has multiple functions, such as flood control, shipping, aquaculture, supporting tourism, and recreation, among which the most important function is of water supply . However, in recent years, the outbreak of cyanobacteria threatens the water ecological security of Taihu Lake. The water temperature ranges from 0 to 35 °C, which is affected by (Liu et al. 2015). The outbreak period of cyanobacteria is from May to September. Affect by the atmospheric temperature, the water temperature is generally 25 ~ 35 °C (Liu et al. 2015;Luo et al. 2019).
Samples were collected from five sites based on routine monitoring points of the Lake − Watershed Science Sub Center, National Earth System Science Data Center, National Science & Technology Infrastructure of China (http:// gre. geoda ta. cn) (Fig. 1A).

Chemical analysis
For the lake water quality observation, electrical conductivity, pH, dissolved oxygen, and water temperature were measured using a multiparameter water quality analyzer (YSI Professional Plus, 6600V2, USA) at the sampling sites. Other factors related to denitrification are the environmental factors including nitrite (NO 2 − ), nitrate (NO 3 − ), and ammonia nitrogen (NH 4 + ). NO 2 − was detected by spectrophotometric method, and NO 3 − was measured by ultraviolet spectrophotometry, and NH 4 + was measured by Nessler's reagent spectrophotometry (Jiang et al. 2019).
The sediment-water content was calculated by drying the samples at 105 °C for 24 h to a constant weight. The dried samples were burnt at 550 °C for 6 h in a muffle furnace to measure the loss on ignition.

Incubation experiment
The intact columnar samples were placed vertically in the laboratory, and the lake water was filled with a syringe along the pipe wall, so that the upper layer of the samples was lake water and the lower layer was sediment, by to ensure that the samples were least disturbed. The  (Liu et al. 2015). To avoid the influence of algal photosynthesis in the sediment and the water on denitrification, the column samples were wrapped with aluminum foil during cultivation. The sediment cores were incubated in the denitrification incubation system, that included five buckets filled with filtered lake water, a peristaltic pump with constant water flow, sealed pistons, inlet pipes, and outlet pipes. The inlet pipe had to be controlled to be lower than the outlet pipe and maintained approximately 1 cm away from the sediment. The filtered lake water was pumped into the sediment cores by a peristaltic pump at a flow rate of 0.78 mL/min, ensuring the overlying water was fully mixed (Fig. 2).
After the pre − culture experiment, 15 NO 3 − was added to each bucket to achieve a final concentration of 100 μmol/L, and then steadily incubated at three temperatures for another 24 h (Veraart et al. 2011). The inlet water of each sampling site was carefully collected into the Labco bottles without bubbles using syringes, and the outlet water directly overflowed into the Labco bottles. Following this, 0.2 mL 50% ZnCl 2 was added to each Labco bottle. Eventually, the content of soluble gas ( 28 N 2 , 29 N 2 , and 30 N 2 ) in the water sample was determined using a membrane interface mass spectrometer (MIMS). In addition, 25 mL of inlet and outlet water samples were collected and filtered through 0.45 μm cellulose acetate membranes to determine the concentration of NO 3 − .

Calculation method of potential N 2 production and denitrification rates
Denitrification in the sediment can occur at the expense of NO 3 − from the water column or from NO 3 − produced within the sediment by nitrification (Steingruber et al. 2001). The main pathway can be identified using the 15 NO 3 − isotope pairing technique (Fig. 3). The dissolved gases used to calculate the denitrification rates were 29 N 2 and 30 N 2 , and their corresponding potential production rates r 29 and r 30 were calculated as follows (Tang et al. 2014;Jiang et al. 2020): where r i (μmol·m −2 ·h −1 ) is the release rate of i N 2 (i = 29, 30), C i , C i0 (μmol·L −1 ) are the concentrations of i N 2 in the inlet and outlet water, respectively, v (mL·min −1 ) is the flow rate of the peristaltic pump, A (m 2 ) is the surface of the incubated sediment, and the time conversion factor is 60.
The denitrification rates of 15 NO 3 − (D 15 ) and unlabeled 14 NO 3 − (D 14 ) can be calculated by the production rate of 29 N 2 (r 29 ) and 30 N 2 (r 30 ), it can normally be expressed as (Steingruber et al. 2001): (1)  (Steingruber et al. 2001;Qin et al. 2020). r 28 , r 29 , and r 30 are the release rates of 28 N 2 , 29 N 2 , and 30 N 2 , respectively. D wtot is the total uncoupled denitrification rate using nitrate from the water col-umn. D w refers to the denitrification rate of nitrate from the water column without tracer addition. D n is the coupled denitrification rate using nitrate from the sediment. D 15 and D 14 are the denitrification rates of 15 NO 3 − and 14 NO 3 − , respectively. D tot is the total denitrification during the incubation experiment The total denitrification rate during the incubation experiment (D tot ) and the total denitrification rate of nitrate from the water column (D wtot ) were determined by the following equations: and where is the 15 NO 3 − abundance during the incubation and is calculated by the following equation: where NO − 3 a and NO − 3 b are the concentrations referred to after and before the addition of 15 NO 3 − tracer addition, respectively.
The nitrate source can be identified by the ratio of D wtot to D tot ( ):

Basic physicochemical parameters of the lake water and sediment
The basic physicochemical parameters of the lake water and sediment at the sampling sites are shown in Table 1. As for the lake water observation, the pH and water temperature of the five sampling sites were approximately 8 and 25 °C, respectively. Therefore, 25 °C was selected as the experimental water temperature for the study of spatial distribution of denitrification rate in Taihu Lake. The dissolved oxygen of s1 and s2 was higher than that of s3, s4, and s5, but the electrical conductivity was evidently lower than that of s3, s4, and s5. In terms of sediment, the average sediment water content of s1, and s2 was nearly 64.35%, and that of the s3, s4, and s5 was approximately 50.24%. The difference in the sediment-water content was not significant. However, the loss on ignition of s3, s4, and s5 was approximately eight times that of s1, and s2 because of the presence of more aquatic plants in the east of Lake Taihu (Ticha et al. 2019).

Denitrification rates and construction of Arrhenius equation
The potential N 2 production and denitrification rates of the five sites at 25 °C were determined by 15 N isotope incubation experiments with MIMS. The results indicated that the denitrification rate had a decreasing trend in the west to the east direction in Lake Taihu. The potential 29 N 2 rates were higher than the 30 N 2 rates at five sites (Fig. 4A). The r 29 of site s1 ranked first, r 29 of s2 and s3 equally ranked second, and r 29 of s5 was only half that of s1. The r 30 of s3 was 23.01 μmol·m −2 ·h −1 , second only to s2, and r 30 of s4 was the smallest (7.54 μmol·m −2 ·h −1 ). The order of D tot was the same as that of r 29 , and the D tot of s1 was 177.39 μmol·m −2 ·h −1 (Fig. 4B). Except for s4 and s5, the D wtot of the other sites was more than 100 μmol·m −2 ·h −1 .
For the purpose of exploring the reason why the total denitrification rate decreases from west to East, the concentrations of NO 2 − , NO 3 − , NH 4 + , and TN in the water at each sampling point of Taihu Lake were detected and analyzed. The concentration of NO 3 − in Lake Taihu was significantly higher than the concentration of NO 2 − and NH 4 + (Fig. 5), and showed the same distinct regional differences as D tot , decreasing from west to east.
For further study, the correlation between the total denitrification rate and the concentration of nitrogen compounds in Taihu Lake was analyzed by SPSS statistical software. The statistical results indicated that there was a more significant positive correlation between NO 3 − and D tot (Table 2). And the reason for the spatial distribution of NO 3 − in Taihu Lake is that the western region of Taihu Lake Basin is the main source of pollutants in Taihu Lake, and the eastern region is the drainage area with good water quality (Zhang 2021).
In addition, the outbreak period of cyanobacteria in Taihu Lake is from May to September, when the water temperature  (Luo et al. 2019). Therefore, the experiments on the effect of water temperature on denitrification were carried out at s1, s2, s3, and s4 points. The results showed that water temperature had a significant effect on denitrification. Specifically, the D tot and D wtot increased with an increase in water temperature when the water temperature ranged from 25 to 30 °C (Fig. 6). The findings of this study were consistent with those of Ma et al. (2008) and Appelboom et al. (2010) on the relationship between water temperature and denitrification. This was mainly due to denitrification reaction could completely take place when the water temperature was between 10 and 30 °C (Liao et al. 2018), and the increase of water temperature would reduce the solubility of oxygen in the water under the appropriate water temperature (Veraart et al. 2011;Zhao et al. 2011) (Table 1). In addition, high temperatures also tended to promote respiration instead of photosynthesis (Minuti et al. 2021), which further reduces dissolved oxygen. As an anaerobic reaction, denitrification was enhanced when the concentration of dissolved oxygen in water decreased. High water temperature not only affects the dissolved oxygen in the water column, but also favors release of nutrients by stimulating microbial mineralization of sediment organic matter, which could increase pore − water nutrient concentrations, or could erode the oxidized microzone at the sediment − water interface by increasing oxygen demand (Holdren and Armstrong 1980;Jiang et al. 2008;Gebremariam et al. 2021;Wang et al. 2021). Thus, the concentration of the substrate increases, and denitrification is promoted, until the nitrate concentration is saturated (Silvennoinen et al. 2008).
On the other hand, the temperature has a significant influence on the growth of microorganisms (Perez-Rodriguez et al. 2017). It was obvious from Fig. 6 that when the water temperature exceeded 30 °C, the appropriate temperature (Liao et al. 2018), D tot decreased with the increase of water temperature. Through experiments, Wang Fig. 4 Potential N 2 production (A) and denitrification rates (B) at five sampling sites (water temperature = 25 °C; all samples, n = 3). r 29 and r 30 are the potential production of 29 N 2 and 30 N 2 , respectively. D tot is the total denitrification during the incubation experiment. D wtot is the total denitrification rate of nitrate from the water column in the sediment Fig. 5 Concentration of nitrogen compounds and D tot at the five sampling sites (water temperature = 25 °C; all samples, n = 3). r is Pearson correlation coefficient between D tot and nitrogen compound (*P < 0.05, significant correlation). D tot is the total denitrification rate during the incubation experiment  (2018) found a response to temperature variation, the microorganisms community presented a little difference after a temperature shock, and the N 2 concentration at 34 °C was lower than that at 25 °C during denitrification. Furthermore, it had been reported that the predominant denitrifying microorganisms belong particularly to the phylum Proteobacteria, when the water temperature was more than 34 °C, the activity of the phylum Proteobacteria in Lake Taihu may decrease, resulting in a decrease in the denitrification rates (Xiao et al. 2015;Zhang et al. 2017). Denitrification rate and water temperature conform to Arrhenius equation (Kaspar 1982;Chi et al. 2004;Ji 2017): where D tot 30 • C (μmol·m −2 ·h −1 ) is the total denitrification rate during the incubation experiment at 30 °C, is the Arrhenius temperature coefficient during denitrification.
Based on the experimental results of denitrification rate in Taihu Lake under the condition of sufficient carbon source, the denitrification Arrhenius equation considering the concentration of water temperature can be preliminarily obtained (Table 2). It can provide a scientific basis for determining the parameters of water environment mathematical models such as the EFDC model or the MIKE model in water quality prediction.

Nitrate source identification of denitrification
As can be seen from Fig. 3 that the total denitrification rate (D tot ) was the sum of coupled denitrification rate using nitrate from the sediment (D n ) and total uncoupled denitrification rate using nitrate from the water column (D wtot ) (Svensson et al. 2001). Therefore, the ratio of D wtot to D tot ( ) can be expressed as: According to formula (9), if > 0.5 , it meant that D wtot > D n , which indicates that the nitrate used for denitrification mainly came from water.
Based on D wtot and D tot of each sampling site, the was obtained. It was evident that the of the five sampling sites were above 0.5, as illustrated in Fig. 7, which showed that the denitrification at the Lake Taihu sediment-water interface primarily occurred at the expense of NO 3 − from the water column. It was further verified that there was a significant positive correlation between denitrification rate and nitrate in overlying water, and nitrate in overlying water was the . D tot is the total denitrificationduring the incubation experiment. D wtot is the total denitrification rate of nitrate from the water column in the sediment Fig. 7 Results of the ratios of D wtot to D tot ( ) in incubation experiments at the sites s1, s2, s3, s4, and s5. D tot is the total denitrification during the incubation experiment. D wtot is the total uncoupled denitrification rate using nitrate from the water column limiting factor of denitrification in Poyang Lake (Tang et al. 2014). Besides, when the water temperature rose from 25 to 30 °C, the increased slightly with the increase in water temperature, and the increase in s2 and s3 was relatively smaller. When the water temperature approached 35 °C, the response at each sampling site was inconsistent, but NO 3 − from the water column was still the main nitrate source of denitrification. In conclusion, the denitrification in the sediment − water interface mainly occurred at the expense of NO 3 − from the water column, and it was less affected by water temperature.

Practical implications of denitrification in shallow eutrophic lakes
Due to human activities, such as large-scale synthetic ammonia and large-scale application of chemical fertilizer, a large amount of exogenous nitrogen enters Taihu Lake, resulting in cyanobacteria bloom and threatening human drinking water and ecological security (Li et al. 2013). And denitrification is considered to be the main biological pathway of nitrogen removal, which is of great significance to reduce lake nitrogen pollution and eutrophication control (Richardson et al. 2004).
Taihu Lake is a shallow lake with an average depth of 1.8 m . The water temperature is strongly affected by temperature and solar radiation, and vertical stratification occurs frequently. The change of lake water temperature will affect the concentration of dissolved oxygen and the thickness of anaerobic layer at the water-sediment interface (Zhao et al. 2011). The change of lake water temperature will also affect the microbial population structure and the activity of denitrifying bacteria, and then change the rate of denitrification, which will eventually change the nitrogen concentration in the lake (Liao et al. 2018).
Moreover, the algal − bacteria system in the water column can form an important niche that favors denitrification during cyanobacteria blooms (Chen et al. 2016. In general, the life of algae is primarily divided into growth and decline periods, and then algae subsides to the bottom of the lake. During the growth period, the oxygen produced by photosynthesis of algae provides an aerobic environment for the nitrifying bacteria attached to the cell mass. This can promote the nitrification of nitrifying bacteria to produce NO 3 − , the substrate for denitrification (Liu et al. 2019). During the decline period, algae are gradually degraded into low molecular weight organic acids (directly available organic carbon) that can be used by denitrifying bacteria (Li et al. 2013).
In general, the concentration of TN in water can characterize the effect of nitrogen removal when the external pollution source is certain. From 2003 to 2006, Lake Taihu maintained a high TN concentration, resulting in cyanobacteria blooms in 2007, following which the cyanobacteria died and were accumulated as sediments. According to the monitoring data, the TN concentration in Lake Taihu gradually decreased from 2008 to 2018 (Fig. 1B). And this further proves that algae contribute to the denitrification during the decay period.
Therefore, the study of denitrification in various regions of Taihu Lake can deepen the understanding of nitrogen cycle process in Taihu Lake and contribute to the effective development of water environment and eutrophication control in Taihu Lake.

Conclusions
In the present study, the effect of water temperature on denitrification was analyzed by 15 N isotope incubation experiments, and the nitrate source of denitrification in Lake Taihu sediment was identified. The results indicated that the denitrification rate showed a decreasing trend in the west to east direction in Lake Taihu. When the water temperature was lower than 30 °C, the rates of the potential N 2 production and denitrification were higher with an increase in water temperature, but when the water temperature was overhigh, denitrification was inhibited. And the NO 3 − from the water column was the main nitrate source of denitrification according to the ratio of D wtot to D tot . These findings, and denitrification Arrhenius equations considering water temperature, and further research on denitrification can provide a scientific basis for determining the parameters of water environment mathematical models in water quality prediction, play an important role in improving the water quality of Lake Taihu, and bear significance for the water environment treatment of other shallow eutrophic lakes in China and abroad.
Author contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Qiuxia Ma, Zhilin Huang, and Min Pang. The first draft of the manuscript was written by Qiuxia Ma and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data availability
The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

Consent to participate Not applicable.
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