Characteristics and Driving Factors of the Aerobic Denitrifying Microbial Community in Baiyangdian Lake, Xiong’an New Area

Here, the ion-exchangeable form of nitrogen (IEF-N), weak-acid extractable form of nitrogen (WAEF-N), strong-alkali extractable form of nitrogen (SAEF-N), strong-oxidant extractable form of nitrogen (SOEF-N), residue nitrogen (Res-N), and total nitrogen (TN) showed spatial differences, and most of the sediment nitrogen fractions exhibited positive correlations in Baiyangdian Lake. High-throughput sequencing analysis revealed that the aerobic denitrification microbial community was composed of proteobacteria (42.04%–99.08%) and unclassified_bacteria (0.92%–57.92%). Moreover, the microbial community exhibited significant differences (R2 = 0.4422, P < 0.05) on the basis of the adonis analysis. T(temperature), Moisture content (MC), sediment total phosphorus (STP), ion-exchangeable form of ammonia (IEF-NH4+-N), weak-acid extractable form of ammonia (WAEF-NH4+-N), weak-acid extractable form of nitrate (WAEF-NO3−-N), and strong-alkali extractable form of ammonia (SAEF-NH4+-N) were the dominant environmental factors and explained 11.1%, 8.2%, 10.7%, 6.9%, 9.3%, 8.1%, 10.5%, 7.5%, and 7% variation, respectively, of the total variation in the microbial community. Furthermore, the network analysis showed that symbiotic relationships accounted for a major percentage of the microbial networks. The keystone aerobic denitrifying bacteria belonged to Comamonas, Rhodobacter, Achromobacter, Aeromonas, Azoarcus, Leptothrix_Burkholderiales, Pseudomonas, Thauera, unclassified_Burkholderiales, and unclassified_bacteria. The composition of the keystone aerobic denitrifying microbial community also exhibited significant differences (R2 = 0.4534, P < 0.05) on the basis of the adonis analysis. T, STP, IEF-NH4+-N, ion-exchangeable form of nitrate (IEF-NO3−-N), WAEF-NO3−-N, SAEF-NH4+-N, and TN were the dominant environmental factors that explained 8.4%, 6.2%, 4.6%, 5.9%, 5.9%, 4.5%, and 9.4% variation, respectively, of the total variation in the keystone aerobic denitrifying microbial community. The systematic investigation could provide a theoretical foundation for the evolution mechanism of the aerobic denitrifying microbial community in Baiyangdian Lake.


Introduction
Excessive nitrogen concentrations present problems for water quality and cause eutrophication [1]. An increase in the nitrogen load degrades water quality. In the past few years, bioremediation has become one of the most promising treatments because of its low maintenance cost, effective performance, and reduced environmental impacts [2,3]. However, there are different oxygen requirements for nitrification and denitrification in the traditional biological nitrogen removal process, which make the nitrogen removal impractical in natural aerobic waters. Fortunately, the first aerobic denitrifying bacterium (Thiosphaera pantotropha) [4] has been isolated, which can achieve nitrogen removal under aerobic conditions. Previous studies have shown that aerobic denitrification occurs in sea sediments [5], Wadden Sea [6], wetland [7], coastal sediments [8] and reservoirs [9,10]. Therefore, recently, many researchers have focused on nitrogen removal with aerobic denitrifying bacteria [11,12].
Currently, some full-scale experiments on bioaugmentation with aerobic denitrification bacteria have been conducted successfully for the remediation of wastewater, urban river, and river sediment. Such as, Duan et al. (2015) explored the nitrogen removal performance of halophilic heterotrophic nitrification-aerobic denitrification SF-16 in saline wastewater [13]; Du et al. (2017) investigated the variation of bacterial community structure in an aerobic denitrification reactor of industrial wastewater [14]; Sun et al. (2018) achieved the bioremediation of sediment through the aerobic denitrification agent in an urban river [1] and evaluated the mechanism of aerobic denitrifying bacteria [15]. Comparison of bioaugmentation in wastewater, urban rivers, and urban sediments has shown that there is limited information on the addition of inoculated bacteria or enhancement of indigenous aerobic denitrification bacteria in situ in natural water ecosystems. To the best of our knowledge, only our previous study not only investigated the effects of stratification and mixing on microbial community structure [16,17]; but also showed that indigenous aerobic denitrification bacteria can be enhanced based on WLA technology in drinking water reservoirs [18][19][20]. Furthermore, it is important to ascertain whether the introduced microbial strains can survive and remain active in natural water ecosystems. In order to isolate efficient aerobic denitrifying bacteria and achieve nitrogen removal through aerobic denitrification in a natural water ecosystem, it was necessary for us to analyze the characteristics and driving factors of an aerobic denitrifying microbial community, especially "keystone species", the dominant environmental factor in a natural water ecosystem. Previous studies have shown that napA is the biomarker of aerobic denitrifying bacteria [9,21]. Therefore, we investigated variations of aerobic denitrifying bacteria through Illumina MiSeq technology based on the aerobic denitrifying functional gene (napA) in Baiyangdian Lake.
In this study, our objectives were to (1) investigate the characteristics of environmental factors; (2) examine the composition of the aerobic denitrifying microbial community and keystone operational taxonomic unit (OTU); (3) evaluate the differences in abundance, diversity, and community structure; (4) analyze the relationship between the microbial community structure and environmental driving factors; and (5) estimate the relative contributions of the environmental driving factors.

Study Area and Sample Collection
Baiyangdian Lake, located in Xiong'an New Area, is the largest freshwater lake in the North China Plain (Figure 1a). Baiyangdian provides an important ecological guarantee for economic development in the surrounding areas. Baiyangdian Lake is divided into the tourist area, living area, natural area, breeding area, and estuary area. Water sampling was performed on January 15, 2019 with a 5-L surface-water sampler (1 m). The surface sediments (depth = 0-4 cm) were collected using a sterilized Petersen stainless steel grab sampler from 14 sites in Baiyangdian Lake (detail information is shown in the Supplementary Material). The samples were transported to our water research laboratory and stored in the dark at 4 • C.

Water and Sediment Quality Analyses
T, DO, pH, ORP, and EC of the water samples were determined using Hydrolab DS5 (Hach Company, USA). According to the standard methods, TN, NO 3 − -N, NO 2 − -N, and NH 4 + -N were measured using DR6000 (Hach Company, USA) [22]. The different forms of transferable nitrogen for sediment were evaluated using the sequential extraction method ( Figure 1b) and more information is shown in the Supplementary Material [23,24].

Water and Sediment Quality Analyses
T, DO, pH, ORP, and EC of the water samples were determined using Hydrolab DS5 (Hach Company, USA). According to the standard methods, TN, NO3 − -N, NO2 − -N, and NH4 + -N were measured using DR6000 (Hach Company, USA) [22]. The different forms of transferable nitrogen for sediment were evaluated using the sequential extraction method (Figure 1b) and more information is shown in the supplementary material [23,24].

Microbial DNA Extraction and MiSeq
The whole DNA of the sediment was extracted using the Soil DNA Kit (Omega, USA). After DNA purification, the extracted DNA was stored at −80 • C for PCR amplification. The extracted DNA was amplified using primer V66F, 5 -TAYTTYYTNHSNAARATHATGTAYGG-3 , and V67R, 5 -DATNGGRTGCATYTCNGCCATRTT-3 , for aerobic denitrifying bacterial napA [9,25]. PCR amplification was performed as follows: 95 • C for 3 min; 40 cycles at 95 • C for 30 s, 55 • C for 30 s, and 72 • C for 45 s; and 72 • C for 10 min [9]. High-throughput sequencing was performed using the Illumina MiSeq platform and standard protocols at Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). Moreover, the sequencing data was deposited in the National Center for Biotechnology Information (NCBI, https://submit.ncbi.nlm.nih.gov/subs/sra/) database with the accession number PRJNA623955.

Microbial Community and Statistical Analysis
On the basis of the OTU data, the richness index, Shannon index, Simpson index, Pielou index, Chao1 index, ACE index, and Good's Coverage were calculated to evaluate the alpha diversity. The differences in the microbial communities were evaluated using principal co-ordinate analysis (PCoA) and permutational MANOVA (adonis) in the vegan package (R.3.5.3). Redundancy analysis (RDA) was used to determine the correlation between the microbial community and predominant environmental factors with variance inflation factor (VIF) < 10 in the vegan package (R.3.5.3). Indicator species analysis was conducted using labdsv package (R.3.5.3). Hierarchical partitioning (HP) analysis was performed to quantitatively evaluate the relative influences of the environmental factors on the aerobic denitrifying bacterial community by using the rdacca.hp package (R.3.5.3) [26].

Overview of the Microbial Communities
After quality trimming, a total of 223754 sequences with an average length of 373 bp were obtained for the 14 sediment samples. MiSeq revealed a total of 1886 OTUs with 97% similarity (Table  S1). The Shannon, Simpson, Pielou, richness, ACE, and Chao1 indexes exhibited significant differences in the estuary area, and those at the BH sample site were minimum values ( .99, respectively. The average coverage for the 14 sample sites was higher than 0.99, which could reflect the real information of the microbial community [29]. PCoA1 and PCoA2 accounted for 90.21% and 3.63%, respectively. The distribution of α-diversity was influenced by PCoA1 ( Figure 3a). Moreover, T, MC, STP, IEF-NH4 + -N, IEF-NO3 − -N, WAEF-NH4 + -N, WAEF-NO3 − -N, SAEF-NH4 + -N, and SAEF-NO3 − -N were important environmental parameters for alpha diversity on the basis of the VIF analysis (VIF < 10; Table S2). The RDA showed that RDA1 and RDA2 accounted for 78.47% and 0.67%, respectively (Figure 3b). RDA1 was mainly influenced for the variation of α-diversity, and T (R = 0.66), MC (R = 0.70), STP (R = 1.00), IEF-NH4 + -N (R = 0.97), and WAEF-NH4 + -N (R = 0.80) were the dominant environmental parameters (Table S2). The relative influences of dominant physicochemical factors on the diversity of the sediment aerobic denitrifying microbial community [30] were evaluated using HP analysis (Figure 3c,d). STP showed

Overview of the Microbial Communities
After quality trimming, a total of 223754 sequences with an average length of 373 bp were obtained for the 14 sediment samples. MiSeq revealed a total of 1886 OTUs with 97% similarity (Table S1). The Shannon, Simpson, Pielou, richness, ACE, and Chao1 indexes exhibited significant differences in the estuary area, and those at the BH sample site were minimum values ( .99, respectively. The average coverage for the 14 sample sites was higher than 0.99, which could reflect the real information of the microbial community [29]. PCoA1 and PCoA2 accounted for 90.21% and 3.63%, respectively. The distribution of α-diversity was influenced by PCoA1 (Figure 3a).  (Table S2). The relative influences of dominant physicochemical factors on the diversity of the sediment aerobic denitrifying microbial community [30] were evaluated using HP analysis (Figure 3c,d). STP showed the greatest

Comparative Analysis and Driving Factors of the Microbial Community
Differences in the composition of the aerobic denitrifying microbial community in the five areas were investigated with PCoA and NMDS analysis. RDA was used to demonstrate the links between the environmental parameters and microbial community. HP analysis was performed to investigate the relative influence of the environmental driving factors on microbial community composition.
For the whole aerobic denitrifying microbial community. The PCoA results showed that PCoA1 and PCoA2 accounted for 42.91% and 17.42%, respectively, of the variability in the microbial community composition (Figure 5a). Moreover, the aerobic denitrifying microbial community composition exhibited significant differences (R 2 = 0.4422, p = 0.012 < 0.05) on the basis of the adonis analysis. The stress result (stress = 0.06 < 0.1) of NMDS showed that the NMDS analysis exhibited good representation ( Figure  S2a). On the basis of the PCoA and NMDS analysis results, samples from the same area clustered together, except for the estuary area; this suggested that the microbial community composition exhibited huge differences. On the basis of VIF (VIF < 10; Table S3), environmental parameters T, MC, STP,  IEF-NH (Figure 5c and Table S3). RDA1 and RDA2 accounted for 29.66% and 15.59%, respectively, of the whole variation in the aerobic denitrifying microbial community, and RDA1 had a major influence on the variation. The samples from the breeding and living areas were located in quarter3; samples from the tourist area, quarter2; and samples from the natural and estuary areas, the same area clustered together, except for the estuary area; this suggested that the microbial community composition exhibited huge differences. On the basis of VIF (VIF < 10; Table S3), environmental parameters T, MC, STP, IEF-NH4 + -N, IEF-NO3 − -N, WAEF-NH4 + -N, WAEF-NO3 − -N, SAEF-NH4 + -N, and SAEF-NO3 − -N were important environmental factors for the whole aerobic denitrifying microbial community (Figure 5c and Table S3). RDA1 and RDA2 accounted for 29.66% and 15.59%, respectively, of the whole variation in the aerobic denitrifying microbial community, and RDA1 had a major influence on the variation. The samples from the breeding and living areas were located in quarter3; samples from the tourist area, quarter2; and samples from the natural and estuary areas,  For the keystone aerobic denitrifying microbial community. The PCoA results showed that PCoA1 and PCoA2 accounted for 45.53% and 32.28%, respectively, of the whole variation in the composition of the keystone aerobic denitrifying microbial community (Figure 5b). Moreover, the  (Table S3). RDA1 and RDA2 accounted for 39.63% and 25.09%, respectively, of the whole variation in the keystone aerobic denitrifying microbial community (Figure 5d). Moreover, the samples from the breeding and living areas were located on the positive side of RDA1, whereas those from the tourist, natural, and estuary areas were located on the negative side of RDA1. T, STP, IEF-NH 4 + -N, .5%, 6.2%, 4.6%, 5.9%, 4.6%, 5.9%, 4.5%, 3.9%, and 9.4% variation of the total variation in the keystone aerobic denitrifying microbial community (Figure 5f).
The correlations between the modules and environmental factors were investigated in Baiyangdian Lake (Figure 6c

Conclusions
This is the first report of the characteristics and driving factors of the aerobic denitrifying microbial community, especially, the "keystone species" and the dominant environment factor in natural water ecosystem through the aerobic denitrification functional gene (napA). In this study, the environmental parameters (IEF-N, WAEF-N, SAEF-N, SOEF-N, Res-N, and TN) exhibited significantly spatial differences among the different functional areas, and most of the sediment nitrogen fractions exhibited positive correlations in Baiyangdian Lake. MiSeq revealed a total of 1886 OTUs identified as proteobacteria (42.04-99.08%), unclassified_bacteria (0.92-57.92%), and Deinococcus-Thermus (0.00-0.25%). The unclassified genus accounted for an important part in Baiyangdian Lake. The RDA and VIF results showed that T, MC, STP, IEF-NH 4 + -N, and WAEF-NH 4 + -N were the dominant environment parameters and explained 11%, 13.6%, 27.6%, 16.3%, and 2.8% variation in α-diversity. Moreover, the microbial community exhibited significantly spatial differences (R 2 = 0.4422, p < 0.05). T, MC, STP, IEF-NO 3 − -N, WAEF-NH 4 + -N, WAEF-NO 3 − -N, and SAEF-NH 4 + -N were the dominant environmental factors and explained the 11.1%, 8.2%, 10.7%, 9.3%, 8.1%, 10.5%, and 7.5% variation of the total variation in the whole aerobic denitrifying microbial community, respectively. The network analysis showed that symbiotic relationships accounted for a major percentage of the microbial networks, and 40.18% nodes belonged to betaproteobacteria. The keystone OTUs belonged to Comamonas, Rhodobacter, Achromobacter, Aeromonas, Azoarcus, Leptothrix_Burkholderiales, Magnetospirillum, Pseudomonas, Sulfuritalea, and Thauera. Furthermore, the composition of the keystone aerobic denitrifying microbial community also exhibited significantly spatial differences (R Acknowledgments: This work was supported by National Natural Science Foundation of China (51909056).

Conflicts of Interest:
The authors declare no conflict of interest.