Comparison among the microbial communities in the lake, lake wetland, and estuary sediments of a plain river network

Abstract Sediment microbial communities from plain river networks exert different effects on pollutant transformation and migration in lake basins. In this study, we examined millions of Illumina reads (16S rRNA gene amplicons) to compare lake, lake wetland, and estuary bacterial communities through a technically consistent approach. Results showed that bacterial communities in the sampled lake sediments had the highest alpha‐diversity (Group B), than in sampled lake wetland sediments and estuary sediments. Proteobacteria was the most abundant (more than 30%) phyla in all the sediments. The lake sediments had more Nitrospirae (1.63%–11.75%) and Acidobacteria (3.46%–10.21%) than the lake wetland and estuary sediments, and estuary sediments had a greater abundance of the phylum Firmicutes (mean of 22.30%). Statistical analysis (LEfSe) revealed that lake wetland sediments contained greater abundances of the class Anaerolineaceae, orders Xanthomonadales, Pseudomonadales, and genera Flavobacterium, Acinetobacter. The lake sediments had a distinct community of diverse primary producers, such as phylum Acidobacteria, order Ignavibacteriales, and families Nitrospiraceae, Hydrogenophilaceae. Total phosphorus and organic matter were the main factors influencing the bacterial communities in sediments from several parts of the lake wetland and river estuary (p < .05). The novel insights into basin pollution control in plain river networks may be obtained from microbial distribution in sediments from different basin regions.

matter transformation and migration. A lake wetland is a landform along the lake region and can be regarded as a filtration system of lake outflow. Bacterial communities in lake wetlands are ubiquitous and play key roles in ecosystem functions, including cycling of biologically active elements (Newton, Jones, Eiler, McMahon, & Bertilsson, 2011; Van der Gucht et al., 2007;Woese, Kandler, & Wheelis, 1990).
Wetland ecosystems are the most biologically diverse around lake basins (Ding et al., 2015;Iasur-Kruh, Hadar, Milstein, Gasith, & Minz, 2010) and provide habitats for biota . As main components of lake basins, estuaries are buffer zones between rivers and lakes or oceans; runoff particles and suspended sediments are transported, stored, and modified in estuaries (Arzayus & Canuel, 2005;Sun, Wang, Wu, Wang, & Li, 2011). Inorganic and organic nutrients are usually stored by sedimentation and released back to the water column, and microbial communities in sediments play important roles in substance export, regeneration, and biochemical cycling in estuaries (Pinckney, Paerl, Tester, & Richardson, 2001;Yokokawa & Nagata, 2010).
In response, the goal of this research was to expand the knowledge of microbial distribution in sediments from different basin regions of a plain river network. This would improve our understanding of microbial function in basin pollution control.
As one of the most densely populated regions in China, Taihu Basin (with a surface area of 1.58 × 10 4 km 2 ) is located in a typical plain river network in the middle-lower Yangtze River plain of eastern China. Rivers and lake wetlands typically exist in Taihu Basin.
Approximately, 20 large-, medium-, and small-sized cities are located around Taihu Lake and have large inflow and outflow rivers, such as Taipu River, Wangyu River, and Dongtiao River, which are connected to Taihu Lake. The northwest part of Taihu Lake generally has small inflow rivers, and most of the pollutants from Yixing City and Changzhou City are discharged into Taihu Lake through these inflow rivers. Taihu Basin also has numerous lake wetlands, which are located in the eastern and southeast parts of Taihu Basin. Numerous lake wetlands exist around the Taipu River and influence the water quality in Jiaxing City. The location of Taihu Lake, the lake wetlands, and estuary rivers are shown in Figure 1.
In this study, we obtained millions of tags from the lake, lake wetland, and estuary sediments obtained from Taihu basin and compared the microbial communities in the sediments. We believe that this study can provide novel insights into basin pollution control in plain river networks.
F I G U R E 1 Sampling site at the study area 2 | E XPERIMENTAL PROCEDURE S

| Sediment collection and characterization
Taihu Lake is the third largest freshwater lake in China and located in the eastern plain of the country (30°55′40″-30°55′40″ N, 119°52′32″-120°36′10″ E) (Xu et al., 2015;Zhu et al., 2013). A total of 65 sediment samples were collected, 32 from the wetland in the southeast part of Taihu Lake (Group A), 27 from the main lake district (Group B), and six from the main river estuaries (Group C). Figure 1 shows the sampling sites in the three regions. The sediment samples were collected in July 2017, and the lake, lake wetland, and estuary sediments were assigned as T, W, and R, respectively. The weights of the collected samples were sufficient for DNA extraction and analysis of physicochemical parameters. After sampling, the sediment samples were placed in sealed plastic bags, stored in a portable ice box, transferred into the laboratory within 24 hr, and stored at −80°C before analysis. Each type of sediments was mixed with deionized water or 0.01 mol L −1 KCl solution in a 1:2.5 (w/v) mixture for pH measurement, and organic matter (OM) content in each sediment was calculated according to the loss on ignition to constant mass (4 hr) at 550°C (Huang et al., 2015). Total nitrogen (TN) and total phosphorus (TP) in sediments were measured with standardized methods and tests (Huang, Chen, Jiang, & Zheng, 2017).
Physicochemical parameters are shown in Figure 2.

| Extraction of genomic DNA
Total genomic DNA was extracted from the sediment samples using a PowerSoil DNA Isolation Kit (Mobio Laboratories Inc., San Diego, CA, USA) according to the manufacturer's protocol. DNA concentration and quality were determined using a NanoDrop Spectrophotometer (Thermo Scientific NanoDrop Lite, CA, USA).
DNA was diluted to 10 ng μl −1 using sterile ultrapure water and stored at −80°C for further analysis.

| PCR amplification
The V4-V5 regions of bacterial 16SrRNA genes were amplified with universal primers 515F (GTGCCAGCMGCCGCGGTAA) and 926R (CCGTCAATTCMTTTRAGTTT). These primers were F I G U R E 2 Physicochemical properties of the sediments from the three regions, Red (a) indicates the lake wetland, green (b) for the lake, and blue (c) for the river estuary. The horizontal lines composing the box from top to bottom represent 3rd quartile, median, and 1st quartile, respectively. The dot represents the average value selected because of their high coverage across phyla (Liu, DeSantis, Andersen, & Knight, 2008;Wang & Qian, 2009;Xiong et al., 2014
Principal component analysis (PCA) was employed to explore and visualize the similarities between sediment samples obtained from three regions based on Bray-Curtis dissimilarity using package F I G U R E 3 Diversity of the sediment samples from the three regions (a for the lake wetland, b for the lake, and c for the river estuary). *p < .05, **p < .01 Ape (R package) (Paradis, Claude, & Strimmer, 2004

| Accession numbers
All of the sequencing data analyzed in this study can be downloaded from the NCBI's Sequence Read Archive using the accession numbers SRP133902 for samples from lake wetland (32 samples), lake (27 samples), and estuary (six samples).

| Diversity indices of bacterial communities in the sediments from different regions
A total of 65 sediment samples were obtained, 32 from the lake wetland (Group A), 27 from the lake (Group B), and six from the estuary (Group C). After tag merge and quality control, 2,822,582 reads were obtained. Although the total number of errors were low, stringent quality controls were set because of the overestimation of alpha-diversity (Fang et al., 2015;Wang et al., 2012). Figure 3 shows

| Bacterial community composition in sediment samples
The  in the sediments from the lake and the estuary (8.35% and 7.89%, respectively). Clostridia was more abundant in the wetland and estuary sediments (4.30% and 11.23%, respectively) than in lake sediments (1.13%). The lake sediments had more Nitrospira (8.53%) than lake wetland and estuary sediments (1.81% and 1.53%, respectively).
Nitrospira and Deltaproteobacteria were more abundant in the lake sediments than in the wetland and estuary sediments (p < .01).
Bacilli, Epsilonproteobacteria, Gemmatimonadetes, Ignavibacteria, Chlorobia, and Phycisphaerae were the other classes detected in the sediment samples from the three regions.
The taxonomic composition at the genus level of the bacterial communities in sediment samples from the three regions is shown in Figure 5. Nitrospira, Thiobacillus, and Sulfuritalea were the most dominant genera in most of the sediment samples from the lake region with relative abundance of more than 4%. The bacterial communities in the wetland sediments had relatively higher abundance of Flavobacterium and Acinetobacter than those in the lake and estuary sediments. The relative abundance of Bacillus, Fusibacter, Lactococcus, and Pseudomonas in the sediment samples from the estuary was more than 6%, which was higher than those in the sediment samples from the two other regions (lower than 1%). Thiobacillus, Anaerolinea, Desulfobulbus, Rhodobacter, and Novosphingobium were the other dominant genera with relatively high abundance in the sediment samples from the three regions. Figure 6 shows the groupings of the sediment samples according to their bacterial community structure. Two PCAs explained 78.3% of the total variation in the microbial community structure.

| Redundancy analysis
RDA was performed on the basis of the abundance at the phylum level and environmental factors (physicochemical parameters). The bacterial communities in the sediments from the lake wetland and estuary were associated with physicochemical parameters, such as OM, pH, DO, and TP content. The bacterial communities in the sediments from several parts of the lake wetland and the river estuary were positively correlated with TP and OM (0.894, p < .05).

| D ISCUSS I ON
Different regions, such as wetland, estuary, and lake side zone, exist in the Taihu Basin. The sediments in these regions are critical to pollutant release and can influence the water quality in Taihu Lake.
The sediment samples from the lake wetland, lake, and river estuary were collected, and the differences among the microbial communities in the three regions were studied.
The diversity indices revealed that the Chao, ACE, Shannon, and Simpson values of the samples from the three regions varied significantly. This result indicated that the bacterial communities in the sediments from the three regions had significant regional differences. Numerous inflow and outflow rivers exist in Taihu Basin.
Rivers 1-4 ( Figure 1) are the main inflow rivers of Taihu Lake, and Rivers 5 and 6 are the outflow rivers. Pollutants entered the lake region through the inflow rivers and were discharged through the outflow rivers. Large pollutants deposition occurred in lake region, which caused the bacterial communities in the sediment from lake region to have higher diversity than those in the sediments from the two other regions (Deng, Cui, Hernandez, & Dumont, 2014;Liu et al., 2009). The bacterial community in the sediment from the lake wetland also had relatively high diversity because of the high amount of pollutant discharge from cities such as Suzhou and Jiaxing (Figure 1).
Although the pollution source exists around the inflow and outflow rivers, most of the pollutants do not easily deposit because of the high-flow velocity. The bacterial diversity in the sediments from the four inflow rivers (Rivers 1-4) was therefore lower than those in sediments from the two other regions (Beardsley, Moss, Malfatti, & Azam, 2011;Wu et al., 2017). In addition, as shown in Figure 2, the sediments from the outflow rivers had low pollutant concentrations, and the sediments from outflow rivers also had the lowest bacterial diversity.
Proteobacteria, a phylum that commonly exists in sediment, is nearly the most abundant phylum detected in samples among the three regions and plays a critical role in degradation and metabolism (Bai et al., 2012;Chaudhry, Rehman, Mishra, Chauhan, & Nautiyal, 2012;Huang & Jiang, 2016). Studies revealed that abundance of Acidobacteria is significantly correlated with pH, and Acidobacteria prefers an environment with low pH of ~5.5 (Jones et al., 2009).
The sediment from the lake had the relatively lowest pH, and Acidobacteria was abundant in the sediment samples from the lake.
The sediment from the lake also had higher Nitrospirae content than those in the sediments from the two other regions. In Taihu Basin, the lake region had high nitrogen concentration (2.62 ± 1.46 mg L −1 , 2013-2014) (Wu, Qin, Yu, Deng, & Zhou, 2016), and the nitrogen release in sediment contributed to the high nitrogen concentration in the lake. Nitrospirae participates in nitrogen cycling and may be one of the main reasons for the high nitrate concentration in the sediments (Huang et al., 2017). The result also indicated that nitrogen might be the factor limiting eutrophication, and our previous study obtained similar results (Huang et al., 2017). The bacterial distribution at the genus level also indicated that the NO 3 -N concentration was higher in the lake than in lake wetland and river estuary because of the participation of Thiobacillus in NO 3 -N metabolism (Bruckner et al., 2013;Li, Zhao, & Wang, 2011). In the sediment from the estuary regions, the bacterial community from outflow river (R5 and R6) had higher abundance of Chloroflexi than that in other sediments F I G U R E 6 PCA of phylum abundance data using Canoco 4.5 (red represents the lake, green represents the lake wetland, and blue represents the estuary) F I G U R E 7 RDA of the bacterial communities as affected by environmental conditions and based on the relative abundance of dominant bacterial phyla (red represents the lake, green represents the lake wetland, and blue represents the estuary) from the inflow rivers. Chloroflexi, a photoautotrophic microbe, may have participated in organic pollutant degradation, thereby possibly causing the low OM content in the outflow rivers. The lake wetlands, located in the southeast part of Taihu basin, are polluted with agricultural pollutants and industrial pollution discharge (Beardsley et al., 2011;Bissett, Bowman, & Burke, 2006) from Jiaxing City (Figure 1).
The region has more organic pollutants than the lake and estuary, especially in sediments. As a result, the lake wetland sediments had high abundance of Gammaproteobacteria, which are usually present in organic-rich sediments (Figure 4b) (Song et al., 2012).
Taihu Lake is an algae-dominated area, and algal blooms typically occur in summer. The algal growth and metabolism are significantly related to the sediment. Previous studies revealed that P is a major limiting factor of eutrophication in Taihu Lake, and the sediments of the algae-dominated area is dominated by Microcystis (Huang et al., 2017;Shao et al., 2011;Wu, Chen, Xu, Liu, & Hahn, 2007). The results of this study revealed that TN concentration in water and sediment significantly influences bacterial community, and Nitrospira (phylum Nitrospirae) and Thiobacillus (phylum Proteobacteria) are significantly related to TN concentration. These two genera were also abundant in the lake sediments ( Figure 5). N forms and concentrations were affected by Nitrospirae, and Thiobacillus directly participated in NO 3 -N metabolism and subsequently influenced TN concentration in the lake (Bruckner et al., 2013;Li et al., 2011).
Therefore, TN concentration in sediment is possibly one of the main factors limiting eutrophication, and the limiting factor of eutrophication in Taihu Lake may be changed from P to N.
In the lake wetland around Taihu Lake, a large amount of pollutants from the towns and cities entered the rivers, which are connected with wetlands, and organic matters were deposited into F I G U R E 8 Cladogram showing the phylogenetic distribution of microbial communities associated with the sediments from the three regions; lineages with LDA values of 3.0 or higher as determined by LEfSe are shown. Differences are represented by the color of the most abundant class. Red (a) indicates lake wetland region, green (b) lake region, and blue (c) river estuary region; yellow represents insignificant difference. The diameter of each circle is proportional to a taxon's abundance. Circles from inner region to outer region represent the phylogenetic levels from domain to genus F I G U R E 9 Indicator microbial groups in the three groups of sediment samples with LDA values higher than 3.0 | 11 of 13 this region, which caused the high OM content in the lake wetland region. Nutrients in this region are frequently transformed from inorganic to organic forms, and may explain the association between Desulfarculaceae (phylum Proteobacteria and class Deltaproteobacteria) and OM. The results indicated that the bacterial communities in sediments from parts of the lake wetland were influenced by TP and OM, and Proteobacteria was significantly positively correlated with TP and OM (p < .05). The inorganic P might therefore transform to organic P, and Desulfarculaceae might be one of the main OM indicators in the lake wetland (Jorgensen, 1982;Pallud & Van Cappellen, 2006).
The sediment samples from R1 to R4 are located in the estuary of inflow rivers. Large amounts of pollutants mixed, and parts of the pollutants were deposited into the surface sediments . The bacterial communities were influenced by the OM, TP, pH, and DO, indicating that the environmental conditions in this region were complex, and the bacterial communities were affected by these factors. In addition, R5 and R6 are the estuaries of outflow rivers. In these regions, flow velocities are high, and pollutants frequently migrated and transformed (Bai et al., 2009).
Pollutants, such as inorganic and organic matters, are hardly deposited into the sediments. OM, DO, and pH, are not the main environmental factors that influence bacterial communities in this region.
In summary, the bacterial communities in the lake, wetland, and estuary sediments varied significantly, and the bacterial community varied according to region. In this study, we comprehensively compared the three regions (i.e., lake, wetland, and estuary) in a plain river network. The lake sediments had the most diverse bacterial communities, followed by the wetland sediments. Proteobacteria was the most abundant of all phyla in the sediments from the three regions. Nitrospirae, Acidobacteria, and Firmicutes were the most abundant phyla in the lake, lake wetland, and estuary sediments, respectively. TN affected the bacterial community in sediments from the lake region, and physicochemical parameters, such as OM, pH, DO, and TP concentrations, influenced the bacterial community in the wetland sediments. TP and OM contents were positively correlated (p < .05) to the distinct bacterial communities in sediments from several parts of the estuary regions.

CO N FLI C T O F I NTE R E S T
None declared.