Seasonal changes in N-cycling functional genes in sediments and their influencing factors in a typical eutrophic shallow lake, China

N-cycling processes mediated by microorganisms are directly linked to the eutrophication of lakes and ecosystem health. Exploring the variation and influencing factors of N-cycling-related genes is of great significance for controlling the eutrophication of lakes. However, seasonal dynamics of genomic information encoding nitrogen (N) cycling in sediments of eutrophic lakes have not yet been clearly addressed. We collected sediments in the Baiyangdian (BYD) Lake in four seasons to explore the dynamic variation of N-cycling functional genes based on a shotgun metagenome sequencing approach and to reveal their key influencing factors. Our results showed that dissimilatory nitrate reduction (DNRA), assimilatory nitrate reduction (ANRA), and denitrification were the dominant N-cycling processes, and the abundance of nirS and amoC were higher than other functional genes by at least one order of magnitude. Functional genes, such as nirS, nirK and amoC, generally showed a consistent decreasing trend from the warming season (i.e., spring, summer, fall) to the cold season (i.e., winter). Furthermore, a significantly higher abundance of nitrification functional genes (e.g., amoB, amoC and hao) in spring and denitrification functional genes (e.g., nirS, norC and nosZ) in fall were observed. N-cycling processes in four seasons were influenced by different dominant environmental factors. Generally, dissolved organic carbon (DOC) or sediment organic matter (SOM), water temperature (T) and antibiotics (e.g., Norfloxacin and ofloxacin) were significantly correlated with N-cycling processes. The findings imply that sediment organic carbon and antibiotics may be potentially key factors influencing N-cycling processes in lake ecosystems, which will provide a reference for nitrogen management in eutrophic lakes.


Introduction
Nitrogen input caused by human activities can greatly affect the processes of the N-cycling of lake ecosystems, leading to the eutrophication of water bodies (Basu et al., 2022;Jiang et al., 2023).It has been proved microorganisms, especially N-cycling functional genes are the key driver of the nitrogen transformation processes in the lakes (Isobe and Ohte, 2014).Therefore, N-cycling functional genes have been given more and more concerns for nitrogen removal of the eutrophic lakes.
N-cycling plays an important role in maintaining the ecological balance of lakes (Isobe and Ohte, 2014).Nitrogen in lakes exists in the form of inorganic nitrogen and organic nitrogen, which is absorbed and assimilated by algae, macrophytes (Wu et al., 2021), benthic animals and other organisms (Wu Y. et al., 2022), and can be converted into biological organic nitrogen (Pajares et al., 2017).After these organisms die, they release a large amount of organic nitrogen and inorganic nitrogen to water and sediments (Li et al., 2012;Wu et al., 2021).In eutrophic lakes, the microbial decomposition of a large number of dead aquatic organisms settling to the bottom of the lakes can cause a lower concentration of dissolved oxygen (Wu et al., 2021), which will lead to the production of ammonia, sulfide and other substances (Hu et al., 2023), having a negative impact on the lake ecosystem health (Wang M. et al., 2023;Wang X. et al., 2023).
The N-cycling processes in sediments mainly involve in nitrogen fixation, nitrification, denitrification, assimilatory nitrate reduction (ANRA), dissimilatory nitrate reduction (DNRA) and anammox (Hu et al., 2023), among which nitrification and denitrification are the most important nitrogen transformation processes.These processes induced by microorganisms can oxidize ammonia nitrogen into nitrate nitrogen, and reduce the bound nitrogen into N 2 O or N 2 back to the atmosphere (Broman et al., 2021).Each pathway of the N-cycling process is completed by the enzyme encoded by the corresponding functional gene using the corresponding substrate catalysis (Broman et al., 2021).However, the abundance and diversity of N-cycling functional genes in lake ecosystems are greatly different due to different water quality (such as water temperature, and nitrogen to phosphorus ratio) (Basu et al., 2022), hydrological conditions (such as lake water exchange cycle) (Stoliker et al., 2016;Li et al., 2021) and seasons (Baumann et al., 2022).Therefore, it is of great significance to explore the changes of N-cycling functional genes in lakes and their influencing factors in different seasons.
Baiyangdian (BYD) Lake (38°43′ ~ 39°02′N, 115°38′ ~ 116°07′E) is the typical eutrophic wetland in North China and has a relatively important geographic position.The BYD Lake water is eutrophicated, accounting for 26.7% of areas "mildly eutrophicated, " accounting for 53.3% of areas "moderately eutrophicated, " and accounting for 20.0% of areas "severely eutrophicated" (Liu et al., 2020;Yao et al., 2023).However, serious eutrophication dominated by seasonal nitrogen and phosphorus pollution occurred due to intense agricultural activities and rural domestic sewage discharge in BYD Lake (Zhao et al., 2011;Cai et al., 2021).Because of the strong exchange between water and surface sediments in shallow lakes, eutrophication might affect the nitrogen cycle in sediments (Shi et al., 2022).The primary objectives of this work were: (1) the key functional genes related to N-cycling have seasonal variability in sediments in the BYD Lake; and (2) some environmental factors can play a key role in regulating the N-cycling process.
All the studied genes of N-cycling (including nitrification, denitrification, nitrogen fixation, DNRA, ANRA, and anammox) were present in the sediments of BYD Lake, although their abundance varied largely among four sampling seasons (Figure 1 and Supplementary Figures S2-S5).According to the results, the functional genes abundance of each N-cycling process followed the order DNRA > ANRA > denitrification > nitrogen fixation > nitrification > anammox (Figure 1A).In general, the abundances of functional genes involved in DNRA, ANRA and denitrification processes were higher than those of other related N-cycling processes, indicating that the sediments in BYD Lake had higher potential of NDRA, ADRA and denitrification.Interestingly, the functional genes of these three N-cycle processes exhibited higher abundance in fall than in other seasons (Figure 1A).
Overall, Figures 1B,C illustrated the seasonal variation of nitrification genes (e.g., amoA, amoB, amoC, and hao) and denitrification genes (e.g., nirk, nirS, norC, and nosZ) processes.Among the nitrification and denitrification genes, the abundance of nirS (from 0.47 to 1.96 log 10 gene copies) and amoC (from 0.14 to 1.95 log 10 gene copies) exceeded the abundance of other functional genes by at least one order of magnitude (Figures 1B,C).Meanwhile, the abundances of such functional genes as nirS, nirK and amoC genes generally showed a consistent decreasing trend from spring, summer, and fall to winter, while, the abundances of nosZ demonstrated an increasing trend, which ranged from 0.31 to 1.68 log 10 gene copies.

Discussion
It is well known that many N-cycling processes are mediated by N-related microorganisms (Isobe and Ohte, 2014).The nitrification and denitrification functional gene abundance could be an indicator of nitrification and denitrification activities, which has been demonstrated by previous studies reporting a positive correlation between them (Jiang et al., 2022).Moreover, the synergistic effect is manifested in a positive correlation of their gene abundance because nitrification can provide sufficient nitrate for denitrification (Jiang et al., 2023).In the current study, a strong correlation was also observed between the abundance of functional genes associated with denitrification (e.g., nirK, nirS, and norB) and nitrification (e.g., amoA, amoB and hao) pathway in spring, fall and winter (p < 0.05).In summer, the supply of nitrate is limited due to higher temperatures and excessive consumption of oxygen by algae and aquatic plants (Zhou et al., 2021), which may be the reason why we did not observe the correlation between functional genes related to nitrification and denitrification processes in summer.
The different responses of the N cycling process to external stresses might be driven by the remodeling of the microbial community, which could be strongly affected by changes in physicalchemical properties (Tan et al., 2022).A previous study has shown that denitrification and DNRA rates were mainly regulated by the abundance of their functional genes (e.g., nirS, nirK and nrfA), followed by environmental factors (e.g., sediment organic carbon) (Jiang et al., 2023).Marshall et al. (2021) also reported a decrease in anammox functional gene abundance in conjunction with the decreasing organic carbon content.Similarly, in the current study, we also found that most of the N-cycling pathways (including denitrification, DNRA, ANRA, and anammox) were significantly correlated with SOM or DOC due to the influence of plant growth and litter in BYD Lake, the contents of SOM and DOC in sediment are higher in summer and fallMoreover, N-cycling functional genes including norC, nirK, narI, and hao showed a significant correlation with DOC (p < 0.05).This could be explained by the fact that organic carbon input can stimulate microbial N-cycling as organic carbon acts as an electron donor for various N-reduction pathways in organotrophic N-reducing reactions, such as denitrification (Baumann et al., 2022).Previous studies have also reported that higher available carbon (DOC) can promote denitrification (Stewart et al., 2013;Morse et al., 2014) due to N-cycling microorganisms can utilize organic carbon for mixed nutrient growth (Jiang et al., 2023).This further illustrates that the denitrification process in the BYD sediment is the dominant process.
Furthermore, our result showed that T (°C) significantly correlated with the denitrification pathway (r ≥ 4, p = 0.01-0.05, Figure 2) in summer and fall.This also confirms that the nitrogen cycle process is a microbial-dominated process and is therefore more (2017) also found that nirK is negatively related to T, furthermore, the elevated temperature will increase denitrification rates (Dai et al., 2020).This highlights the importance of temperature as one of the main factors influencing the functional genes related to N-cycling in lakes (Yuan et al., 2023).Therefore, the impact of seasonal changes on N-cycling triggering the retention and emission of nitrogen in the lake should be paid more attention by the management department.
Previous studies have also reported that antibiotic pollution could alter the N-cycling process (Wu J. et al., 2022).For example, sulfadiazine inhibits functional genes related to denitrification and anaerobic ammonium oxidation in sediments (Wang M. et al., 2023;Wang X. et al., 2023).As well as, nitrifier-denitrification rates were inhibited by sulfamethoxazole (Chen et al., 2022).Remarkably, in the current study, N-cycling pathways significantly correlated with antibiotics.For instance, denitrification exhibited a significant correlation with NOR in spring (r ≥ 4, p = 0.01-0.05, Figure 2), and OFL in summer (r ≥ 4, p = 0.01-0.05, Figure 2).Nitrification was significantly correlated with SPD (r ≥ 4, p = 0.01-0.05, Figure 2).Anammox had a significant correlation with TC in spring and fall, and NOR in fall (r ≥ 4, p = 0.01-0.05, Figure 2).Our previous study (Zhang et al., 2023) found that NOR and OFL was the main antibiotics in BYD lake sediments, indicating that more attention should be paid to the effect of antibiotics on the N-cycling in the future.Consequently, more concerns should be given to antibiotics pollution in N-cycling studies in eutrophic water bodies.