Spatial distribution characteristics of typical pathogens in sedimentsAnalysis of microbial community structure in surface sediments

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 pathogens that had certain similarities and differences. Clostridium sensu stricto, a potential pathogen which is widely distributed in soil, sludge, human and animal intestines, etc., had the greatest average abundance in each sediment sample (18.975.80%); it peaked in the central area (4#) at 30.03%, and its abundance in the point-source pollution area (18#) was lowest at 10.15%. The high abundance of Clostridium sensu stricto means that the sediments in the Shahe Reservoir are in an anaerobic environment. Acinetobacter is a pathogen widely distributed in soil and water. Its average abundance in each sediment sample was 7.904.51%. The Beisha River channel (15#) had the highest abundance (18.39%). The abundance of the point-source pollution area (18#) was the lowest at 2.26%.

(a) Heat map of the top 10 genera in each sample based on the reads (log2 transformed). (b) Horizontal distribution of E. coli and Enterococcus in Shahe Reservoir sediments. (The Fig.3a has been prepared using Heml 1.0 software).

Romboutsia, a common intestinal pathogen, had a high average abundance in the sediments of Shahe Reservoir (6.552.00%), but its distribution was completely opposite to the distribution of Acinetobacter. Romboutsia had its highest abundance (9.40%) in the point-source pollution area (18#) and its lowest abundance (2.87%) in the river channel (15#).

The average abundance of Povalibacter and Trichococcus species reached 5.342.24% and 3.942.22%, respectively, but they also showed low abundance at the point-source pollution area (18#), whereas abundance at sampling points in other areas was high. The highest abundance for Povalibacter and Trichococcus was 8.16% (downstream of the reservoir, 6#) and 7.71% (upstream of the reservoir, 7#), respectively, and the lowest was 0.93% (point-source pollution area, 18#) and 1.25% (channel, 15# sampling point).

The average abundances of Sporacetigenium, Subdivision3_genera_incertae_sedis, Clostridium XI, Litorilinea, Smithella, and Thermomarinilinea were all above 2.00%: their maximum values were 5.99% (15#), 3.50% (3#), 13.06% (18#), 5.25% (14#), 7.56% (18#), and 4.22% (12#), respectively, while the minimum values were1.43% (4#), 1.46% (14#), 0.59%(4#), 1.54% (3#), 0.97% (15#), and 0.83% (18#). Thus, different microorganisms showed certain individual differences, but generally they had relatively low population abundance in the reservoir core area and point-source pollution area, and displayed high population abundance in different regions of Shahe Reservoir.

The results of this study (Fig.2) showed significant differences in the distribution of E. coli and Enterococcus in different regions. The horizontal distribution range of E. coli content was between 1.50106 and 1.56108 copiesg1, with an average value of 2.691074.71107 copiesg1. The highest value was at the 12# sampling point in the lower reaches of Shahe Reservoir (1.56108 copiesg1), which was one to two orders of magnitude greater than at other regions. The average content of E. coli in the downstream area (6#, 12#) reached 7.971077.68107 copiesg1, followed by the point-source pollution areas (18#, 6.76107 copiesg1), which were higher than the central area (3#, 4#, 5.151062.26106 copiesg1) and upstream (1#, 7#, 1.111072.84106 copiesg1), while the average content in the river channel (14#, 15#, 16#) was relatively low (3.261061.75106 copiesg1).

The horizontal distribution range of ENT content was between 3.56108 and 3.74109 copiesg1, with an average value of 1.821091.23109 copiesg1. The highest value appeared in the upper reaches of Shahe Reservoir at the 7# sampling point (3.74109 copiesg1) and was about an order of magnitude higher than at the sampling points in other regions. The average content of ENT in the central area (2.601091.15109 copiesg1) was relatively high, followed by the point-source pollution area (2.46109 copiesg1), downstream (2.171099.19108 copiesg1), upstream (2.171091.47109 copiesg1), and finally in the Shahe Reservoir (6.131081.96108 copiesg1), where the content was relatively low.

The above results show that the content of ENT in the surface sediments of Shahe Reservoir was about two orders of magnitude higher than that of E. coli. Further, the content of E. coli in the 12# sampling point downstream of the reservoir and the content of ENT in the surface sediments of the upstream area of the reservoir are higher than in other areas.

Based on the results of OTU classification to the genus level, a heat map (Fig.3a) was constructed to study the vertical distribution characteristics of the microbial community structure in the sediments of Shahe Reservoir. A large number of potential pathogenic bacteria were found, which displayed certain commonalities (between different depths of the same sediment column) as well as differences (between different sediment columns).

(a) Heat map of the top 10 genera in each sample based on the reads (log2 transformed). (b) Vertical distribution of E. coli and Enterococcus in Shahe Reservoir sediments. (The Fig.3a has been prepared using Heml 1.0 software).

Sulfuricurvum, a genus of potential pathogens that is widely distributed and commonly found in soil and sludge, had the highest average abundance (18.77%16.71%) in the sediment column samples. Among these, in the 3# sediment column from the reservoir center it was most abundant (44.52%) at the depth of 34cm, but at its lowest (0.56%) at 2cm from the surface.

Arcobacter is a genus of pathogenic bacteria commonly found in humans, animals, and the environment. A. cryaerophilus can cause inflammation of the human intestines. The symptoms of A. butzleri infection include abdominal pain, nausea, vomiting, and diarrhea caused by fever. The average abundance of Arcobacter found in each sediment column sample was relatively high (8.35%18.09%). However, contrary to the vertical distribution of Sulfuricurvum in the 3# sediment column from Shahe Reservoir center, Arcobacter had its highest abundance (59.13%) at 2cm. The abundance was lowest (0.07%) at 1422cm of the 3# sediment column.

Thiobacillus had a relatively high abundance in the vertical distribution of sediments in Shahe Reservoir (6.59%4.66%). The average abundance of Clostridium sensu stricto, Lactobacillus, and Conexibacter in the vertical distribution of the sedimentary column were above 3.00%, with the highest abundance values of Clostridium sensu stricto and Lactobacillus appearing in the Nansha River channel 16# sediment column at 1218cm (respectively, 13.75%, 10.70%), and the highest value of Conexibacter abundance at 412cm (10.41) of the 3# sediment column from the reservoir center. The minimum values of the three species were in the surface 2cm of the 3# sediment column (0.56%, 0.26%, 0.06%).

The vertical distributions of E. coli and ENT in the sediments of Shahe Reservoir are shown in Fig.3b. The content of E. coli in the 3# sediment column from the reservoir center, the 14# sediment column from Beisha River, and the 16# sediment column from Nansha River ranged, respectively, between 1.76104 and 2.95104 copiesg1, 1.04103 and 2.97102 copiesg1, and 3.34104 and 6.56102 copiesg1. The mean values of each were 2.251044.19105 copiesg1, 1.551021.19102 copiesg1 and 2.481022.89102 copiesg1. Thus, it was found that the content of E. coli in the 16# column from the Nansha River channel was about 1.6 times that of the 14# column from Beisha River channel, and two orders of magnitude higher than that of the 3# column from the reservoir center. It is worth noting that the E. coli in all three sediment columns showed a gradual increasing trend with the increase of depth. The content of the three columns at 2cm from the surface was relatively low (1.76104, 1.53103 and 3.34104 copiesg1), but became higher at about 1525cm (2.10104, 2.97102 and 6.56102 copiesg1).

There was little difference between the vertical distributions of ENT and E. coli. The content of ENT in the 3#column, 14#column, and 16#column ranged, respectively, between 2.44103 and 2.13102 copiesg1, 1.48102 and 1.14101 copiesg1, and 1.32102 and 5.12102 copiesg1. The average values were, respectively, 1.381026.73103 copiesg1, 6.151024.00102 copiesg1, and 2.991021.53102 copiesg1. Unlike E. coli, the content of ENT in the Beisha River 14# sediment column was relatively high, about 2.06 times that of the Nansha River 16# column and 4.45 times that of the reservoir center 3# column. The vertical distribution was the same as for E. coli. The content of ENT in the 3# column from the reservoir center, the 14# Beisha River column, and the 16# Nansha River column were all lower at 2cm from the surface of the sediment (respectively, 2.44103 copiesg1, 1.48102 copiesg1 and 1.32102 copiesg1), and higher at about 1525cm (2.13102 copiesg 1, 1.14101 copiesg1 and 4.44102 copiesg1).

As shown in Fig.4a, the TN content of the surface sediments (020cm) from the Shahe Reservoir ranged from 610.00 to 5420.00mgkg1, with an average value of 2759.441450.54mgkg1. The content of TN in the sediments from the point-source pollution area and downstream of the reservoir was significantly greater than that in the core area of the reservoir, the river channel, and upstream of the reservoir. Shahe Reservoir is long and narrow. The reclaimed water (about 80,000 m3d1) flows into the Beisha reservoir (near the 13#sampling point), and the downstream of the reservoir is intercepted by a sluice dam. Therefore, flow velocity at the mouth and upstream of the reservoir is higher than at the middle and downstream. Although pollutants in the reservoir water body may have a tendency to gradually decrease from upstream to downstream, the particulate pollutants are more likely to be deposited in the downstream of the reservoir than at the entrance and upstream. Under normal water depth conditions, the particulate pollutants are at the mudwater interface and in deep water. While the self-purification rate of the area may not be high, the sedimentation is more obvious. Therefore, the phenomenon that pollutants in the sediments gradually accumulate from the upstream to the downstream of the reservoir is manifested in the TN content in the sediment, which increased sequentially from the upstream of the reservoir (1898.001047.54mgkg1) through the central area (2996.671405.13mgkg1) to the lower reaches (4500.00920.00mgkg1). The highest value of TN in the sediments of the reservoir area was located at the 12# sampling point (5420.00mgkg1) downstream of the reservoir. The content of TN at sampling points 4# and 5# in the center area was relatively low (840.00mgkg1, 1950.00mgkg1), due to the installation of aeration facilities in the overlying water body. Wu Bi17, Li Jinrong18, and others found that increased dissolved oxygen will promote the release of nitrogen and phosphorus nutrients from the sediments and thus reduce their content.

Source:WGS 1984).

(a) Horizontal characteristics of TN and TP in the sediments. (b) Vertical characteristics of TN and TP in the sediments. (The figure was created by using ArcGIS software 10.2;

The content of TP in the surface sediments of Shahe Reservoir ranged from 740.00 to 2440.00mgkg1, with an average value of 1444.33395.55mgkg1. As with the horizontal distribution of TN, TP also increased from the upper reaches of the reservoir (1264.00104.61mgkg1) through the central area of the reservoir (1340.00332.47mgkg1) to the lower reaches of the reservoir (1750.0010.00mgkg1). In the river course it was 1605.00522.61mgkg1. Point-source pollution areas were slightly higher (2150.00mgkg1) than the surface sediments of the reservoir area. This is because the point-source pollution area of Shahe Reservoir is mostly near the urbanrural junction, with a high pollution-to-radius ratio.

The sources of pollutants are mostly domestic sewage, surface runoff, and pipeline sediments, and the proportion of phosphorus pollutants is often high. For example, Li Siyuan19 found that 1130% NH4+-N, 1835% TN, and 1947% TP of the point-source pollution in the old city of Changzhou was from domestic sewage, while 2346% NH4+-N, 4356% TN, and 4262% TP came from pipeline sediments.

After sewage and pipeline sediments entered Shahe Reservoir, the sediments and interstitial waters of the point-source pollution area showed significantly higher levels of nutrients such as nitrogen and phosphorus than were found in the river course, the upper reaches of the reservoir, the core area, and the lower reaches of the reservoir. This may be because the sediments of Shahe Reservoir are an important source of nitrogen and phosphorus nutrients. The content of TP in the surface sediments of the 16# sampling point (1220.00mgkg1) and 17# sampling point (1100.00mgkg1) of the Nansha River channel were low, due to the amount of algae and aquatic plants in the overlying water of the river. A large amount of phosphorus released by sediments is used by algae and aquatic plants20. At the same time, the river flow is faster, which reduces the phosphorus content in the sediments. The difference in the spatial distribution characteristics of TP in the surface sediments of Shahe Reservoir, in addition to hydraulic factors, may be related to the chemical environmental effects of different locations in the reservoir area21 and different microbial effects22.

Figure4b shows the vertical change characteristics of TN and TP in the sediments of the Nansha River and Beisha River (near the point-source pollution area) and in the reservoir core area. The TN content of the 3# sediment column (042cm) in the central area, the 14# sediment column (030cm) near the point-source pollution area, and the 16# sediment column (026cm) of the Nansha River channel ranged, respectively, between 1210.00 and 9540.00mgkg1, 1400.00 and 6640.00mgkg1, and 1100.00 and 5480.00mgkg1. The mean values were 4230.952643.50mgkg1, 3485.331420.50mgkg1, and 2723.081456.81mgkg1. For the same sampling points, the content range of TP ranged, respectively, between 1040.00 and 2890.00mgkg1, 1110.00 and 3550.00mgkg1, and 670.00 and 1630.00mgkg1. The average values of TP were 1726.14561.22mgkg1, 2100.67617.59mgkg1, and 1161.54287.40mgkg1.

The vertical distributions of TN and TP in the sediments showed a large change in the content of the surface layer and a small difference in the lower layer. From 10cm, the content of TN and TP in the surface layer had an increasing trend. The distribution of TN and TP presented a three-stage feature of decrease-increasedecrease, with an enrichment layer at 1020cm. This may be due to the continuous increase of phosphorus load in the lake caused by human activity and industrial production in the upper reaches of Shahe Reservoir23. Zhang Wei et al.12 found that the water content of sediments below 30cm in Shahe Reservoir was relatively stable and, based on the time of construction of the dam(1960), the sediment thickness was estimated to be about 30cm, with a linear sediment deposition rate of 0.60cmyr1. This is consistent with the analysis results of this study. It can be seen from Fig.4b that the content of TN and TP below 30cm in the 3# column in the central area is in a stable state, and the 030cm is mainly the sediment produced by external pollution since the construction of the reservoir.

The Pearson correlation analysis of the abundance of E. coli and ENT in the surface sediments and the horizontal distribution of TN and TP (Fig.5) showed a significant positive correlation between E. coli and both TN (r=0.638, P<0.05) and TP (r=0.755, P<0.05); however, ENT and TN (r=0.131, P>0.05) were not significantly correlated, although there was a significant positive correlation with ENT and TP (r=0.752, P<0.05).

Pearson correlation analysis between pathogenic bacteria and TN and TP in the sediment. (The figure has been prepared using IBM SPSS 25.0 software).

The Pearson correlation analysis of the relative abundance of E. coli and ENT in the sediments of Shahe Reservoir and the vertical distribution of TN and TP is shown in Table 2. There was a significant negative correlation between the E. coli in the 3# sediment column in the center area and TN (P<0.05) and also TP (P<0.05); the E. coli in the 14# sediment column had a significant negative correlation with TP (P<0.05), but the correlation with TN was not significant (P>0.05); in the 16# sediment column, the correlations between E. coli and both TN and TP were not significant (P>0.05).

The nutrients in the water body will promote the growth and reproduction of aquatic plants and phytoplankton, and at the same time, phytoplankton will produce a large amount of organic matter through photosynthesis24. In addition, when the number of phytoplankton increases, the food intake of zooplankton also increases, and the excrement increases, which makes the organic matter in the water body increase. The growth and reproduction of aquatic plants also provides a suitable environment for the growth of microorganisms. Scholars such as Wang Mi pointed out in related investigations that TP and TN are environmental factors that affect phytoplankton in the North Canal; The study by Guo Feifei25 et al. on Hubei Jinshahe Reservoir showed that PO43-P affects the structure of microbial communities. Main environmental factors. Therefore, in addition to controlling microorganisms, the reservoir area should also strengthen the control of nutrients.

The correlation between ENT and the vertical distribution of TN and TP in the sediments of Shahe Reservoir was significantly different from that of E. coli. The main difference was that the correlations between ENT and TN and TP in the 3# column of the center area were not significant; Beisha River channel ENT in the 14# sediment column had a significant negative correlation with TP (P<0.05), but the correlation with TN was not significant (P>0.05); in the Nansha River channel 16# column, ENT had a significant negative correlation with TP (P<0.05), and with TN a very significant negative correlation (P<0.01).

It is worth noting that the Pearson correlation of the vertical distribution of E. coli, ENT, TN, and TP in the sediments of Shahe Reservoir was significantly different from correlation results of the horizontal distribution, mainly manifested in the significant negative correlations. The reason may be that changes in environmental conditions (pH more acid or alkali, higher water temperature, increased dissolved oxygen, stronger hydrodynamic conditions, etc.) release nutrients such as nitrogen and phosphorus26 and then, as a result, the content of TN and TP in the surface layer of the sediment becomes higher and the content in the deep layer becomes lower. The migration and fate conditions of E. coli and ENT are different from those of nitrogen and phosphorus, as they are mainly affected by factors such as strain type, bacterial solution concentration, ionic strength, ion type, median particle size, pore flow rate, etc27. As a result, the Pearson correlation between E. coli and ENT in the sediments of Shahe Reservoir and the vertical distribution of TN and TP showed a negative correlation.

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The spatial distribution characteristics of typical pathogens and nitrogen and phosphorus in the sediments of Shahe Reservoir and their relationships...