The spatial distribution characteristics of typical pathogens and nitrogen and phosphorus in the sediments of Shahe Reservoir and their relationships Wen Sun1,2,3,4, Ke Yang1,2,3,4, Risheng Li1,2,3,4, Tianqing Chen1,2,3,4, Longfei Xia1,2,3,4, Zhao Wang1,2,3,4 & Xubo Sun1,2,3,4 Scientific Reports 11, Article number: 21745 (2021) Cite this article Environmental impactPollution remediation Using samples collected in Shahe Reservoir in the upper North Canal in China, this research analyzes the structure of a microorganism group in sediment and the absolute abundance of two typical pathogenic bacteria (Escherichia coli and Enterococcus), and their relationship with environmental factors including total nitrogen (TN) and total phosphorus (TP). The study of samples collected from the surface (0–20 cm) and sediment cores shows that the absolute abundance of E. coli in horizontal distribution in the sediment is highest in downstream of the reservoir and point source pollution area. In vertical distribution, the absolute gene expression level of the two pathogenic bacteria in the sediment tends to decrease with increasing depth, although its highest value at 10–30 cm depth. The relative abundance the two pathogenic bacteria is much greater in the sediment of Shahe Reservoir with the structure of horizontal groups including Clortridium sensu stricto, unclassified Anaeroineaceae, and Povalibacter, while Anaeroineaceae is much more abundant in the group structure of the vertical distribution. Pearson correlation analysis suggests positive correlation in horizontal distribution for E. coli and TN and TP (P < 0.05) and for Enterococcus and TP (P < 0.05). The results clearly show that the amount of pathogenic bacteria in the sediment in Shahe Reservoir is most likely due to water eutrophication. The contamination of surface water bodies by pathogenic bacteria poses a huge potential threat to human health. The seven major water systems in China have all been contaminated by pathogenic bacteria to varying degrees1. Pathogens are widely distributed in both surface water and sediments, while the sediments themselves can provide various protections for pathogens (reducing ultraviolet radiation, providing nutrients, etc.)2. Thus, sediments can aid the long-term survival and growth of pathogenic bacteria in water bodies, acting as both the 'source' and 'sink'3. Studies have shown that those pathogenic bacteria that are widely enriched in surface water sediments include Escherichia coli (E. coli), fecal coliform (FC), Enterococcus (ENT), total coliform (TC), Campylobacter and Salmonella, etc., and that disturbances to the sediment or water can cause the further release and adsorption of pathogenic bacteria4,5,6,7. Some scholars have reported on the investigation of microbial diversity in the water system of the North Canal and the evaluation of river health. For example, Wang Mi et al.8 investigated the microorganisms of the North Canal water system, and the results of water quality evaluation based on the microorganisms showed that the North Canal water system was in a state of moderate pollution; Gu Xiaoyun et al.9 developed the ecosystem of the North Canal (Beijing section) The health assessment pointed out that the health status of the North Canal ecosystem is generally poor. In addition, a large number of studies have shown that nitrogen and phosphorus in sediments are fundamental and crucial factors affecting the eutrophication of water bodies. However, the relationship between pathogenic bacteria and nitrogen and phosphorus in surface water sediments is not yet clear.The current strict implementation of the Water Pollution Prevention and Control Action Plan in China has focused more on organic pollution and eutrophication, but the prevention and control of pathogenic microorganism pollution in rivers need to be further strengthened10 to be more conducive to public health safety management of river basins11. For example, in addition to the problem of eutrophication in the Wenyu section of the North Canal, the upstream and downstream microbic pollution is severe, with the surface water concentration of FC on average exceeding the Class V water quality standard (GB3838-2002) by two orders of magnitude. At present, related researches mostly focus on the relationship between nitrogen and phosphorus nutrients and algae or the relationship between microbes and algae. However, there are few studies on the relationship between nitrogen and phosphorus nutrients and pathogenic microorganisms. Therefore, this study chose Shahe Reservoir in the upper reaches of the North Canal as the research site to investigate the spatial distribution characteristics of microbic communities and nitrogen and phosphorus in sediments, and selected the characteristic pathogenic bacteria E. coli and ENT for this analysis. The relationships between these typical pathogenic bacteria and nitrogen and phosphorus are expected to provide a scientific basis for the treatment of river pathogen pollution and eutrophication.Shahe Reservoir is an important node located in the source area of the North Canal (Fig. 1). The drainage area of Shahe Reservoir is about 1125 km2, of which the mountain area accounts for about 75% 12. The North Canal is an important drainage channel for Beijing. From 1999 to 2005, the average annual sewage storage volume accounted for about 55% of the total incoming water13. The three main tributaries that merge into the Shahe Reservoir, Beisha River, Dongsha River, and Nansha River, have drainage areas of 597 km2, 265 km2, and 263 km2, respectively14. The Shahe Reservoir is a river-type reservoir controlled by the Shahe Sluice, and was built in 1960. Total area around the reservoir is about 1.8 km2. The annual average water level is about 36 m, total storage capacity is 20.45 million m3, and the historical daily mean outflow is about 125,000 m3 per day. The hydraulic retention time is 69–110 days, and the fluidity is poor during the water storage period. During the annual flood season (June–September), the Shahe Reservoir will open the gate and release the water. At this time, the water level will drop rapidly and the flow velocity will increase significantly15.Figure 1Source:WGS 1984).Arrangement and zoning map of sediment and interstitial water sampling points at Shahe Reservoir. (The figure was created by Sun Wen16 and modified using ArcGIS software 10.2;Sample collection and processingBased on their topographical characteristics, 18 sediment sampling points were set up within the Shahe Reservoir study area (Fig. 1). In November 2017, a Peterson mud harvester was used to collect 0–20 cm surface sediment to analyze the horizontal distribution characteristics of nutrients and pathogens. Three columnar sediment samples were collected at sampling points 3#, 14#, and 16# using a mud core sampler to analyze the vertical distribution characteristics of nutrients and pathogens. (r = 50 mm, h = 60 cm).The collected surface sediment was protected from light, stored at low temperature, and then brought back to the laboratory. The sediment columns were layered at 2 cm intervals, and the layered samples and sediment surface samples were freeze-dried (Model FD-1A-50 freeze dryer, Beijing Boyikang Experimental Instrument Co., Ltd.), crushed with a glass rod to remove impurities such as gravel, shells, and animal and plant residues, ground with a mortar, and passed through a 100-mesh sieve before analysis. Meanwhile, the samples obtained by the Peterson mud harvester were mixed and put into a 50 mL centrifuge tube, centrifuged at 4000 rpm for 20 min to obtain interstitial water, and stored at − 4 °C.Take a fresh sample of the sediment and use the drying method to determine the moisture content and organic matter (expressed as loss on ignition, LOI). An elemental analyzer (Vario MAX cube, Elementar) was used to determine the total nitrogen (TN) content value in the sediment. Total Phosphorus (TP) in the sediment was extracted by the SMT (Standards, Measurements and Testing) method developed under the framework of the European Standards and Testing Committee. The sediment samples were burned at 450 °C and oscillated at room temperature with 3.5 mol/L HCl After 16 h, the molybdenum antimony spectrophotometric method was used to determine the TP content in the extract.In this study, the typical pathogenic bacteria Escherichia coli and Enterococcus were selected as indicator bacteria for quantitative analysis of qPCR, and the gene copy number (DNA copies·g−1) was used to express its corresponding content in the sediment, and the relative abundance was standardized by 16S rRNA.DNA extractionthe sediment sample is freeze-dried and weighed 0.1 g In a 2 mL lysis tube, use the FastDNA Spin Kit for Soil kit (MPbio, USA), and extract DNA according to the instructions of the kit.Microbial community structure analysisBased on high-throughput sequencing to determine the 16S rRNA V4 region PCR product gene sequence, and analyze the microbial community structure in each sample. The PCR primer used is 515F/806R, and the barcode sequence is added before the forward primer to distinguish the PCR products of different samples. Each sample is repeated 3 times for PCR and mixed, and then the PCR products are recovered from different samples. The PCR products were mixed in equal amounts, library construction and sequencing; library construction and sequencing were completed by Sangong Bioengineering (Shanghai) Co., Ltd. The sequencing platform was Illumina MiseqTM. For Miseq paired-end sequencing data, first remove the primer linker sequence (TGGAATTTCTCTGGGTGCCCAAGGAACTC), and then according to the overlap relationship between PE reads, the paired reads are spliced into a sequence and distinguished according to each sequence of the samples, and then the samples are distinguished according to the sequence. Sample data, and finally perform quality control filtering on the quality of each sample data to obtain valid data for each sample.Quantitative PCR (qPCR) analysisThe main reagents used for quantitative PCR (qPCR) analysis in this study were SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (TAKARA) and RNase-free Water (Ambion). The qPCR analysis was carried out on a micro ultraviolet spectrophotometer (Nanodrop 2000) and a fluorescent quantitative PCR instrument (StepOne Plus). The amplification efficiencies of the target gene fragments of Enterococcus and E. coli were 99.54% and 97.82%, respectively. The specific primer sequences and mechanisms are shown in Table 1.Table 1 Primers and their mechanisms used in this study.Analysis of microbial community structure and typical pathogensBased on metagenomic classification and sequencing, the PCR products of 16S rRNA V4 regions were determined, and the microbial community structure in each sample was analyzed. The PCR primer used was 515F/806R, and the barcode sequence was added before the forward primer to distinguish the PCR products of different samples. The PCR for each sample was repeated three times before they were mixed. For this, the PCR products were recovered using gel, and the PCR products from different samples were mixed in equal amounts for library construction and sequencing; library construction and sequencing were completed by related sequencing companies, and the sequencing platform was Illumina Miseq × 250. For Miseq paired-end sequencing data, the primer adapter sequence (TGGAATTCTCGGGTGCCAAGGAACTC) needed to be removed first, and then the paired reads were merged into a sequence according to the overlap relationship between paired-end reads. Samples were then identified and distinguished according to the barcode tag sequence. Finally, quality control filtering was performed on the samples to ensure valid data for each sample.Following guidance on relevant standards for pathogens in surface waters from the United States Environmental Protection Agency, the European Union, and the World Health Organization, this study selected the typical pathogens E. coli and ENT for analysis, using their gene copy numbers (copies g−1 ) to represent their corresponding content in the sediment, and their proportion (%) in 16S rRNA to represent their abundance.Data processing and analysis methodsSPSS 25.0 software was used to analyze the correlation between the pathogens and total nitrogen (TN) and total phosphorus (TP) in sediments. The horizontal spatial distribution characteristics of nutrients in the sediments were analyzed using the ArcGIS 10.2 software package, The projection coordinate system was selected as "Chinese Albers Projection", and the analysis method was selected as the inverse distance weighting interpolation method. The vertical spatial distribution of nutrients in the sediment and the absolute content of pathogens (copies g−1) were analyzed by Origin 2017. The heat map of the microbial community structure in the sediment was constructed using Heml 1.0 (http://hemi.biocuckoo.org/down.php) (Wankun et al., 2014). The R language ade4 package was used to perform noise reduction analysis on operational taxonomic units (OTUs) in the community structure. The average abundance of OTUs in all samples was required to be higher than 0.01%. The OTUs after noise reduction analysis were used for subsequent analysis.It can be seen from Fig. 2 that the microbial community structures of the various surface sediments of Shahe Reservoir contained a large number of potential pathoge
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The spatial distribution characteristics of typical pathogens and nitrogen and phosphorus in the sediments of Shahe Reservoir and their relationships
