The operation of large-scale water conservancy projects can change the natural hydrological cycles and sediment translocation processes in downstream linked lakes [1
]. The damming of rivers has had a significant global impact on natural water resources [3
], as impoundment of dams can affect the water environments, i.e.
, physical, chemical, and biological characteristics, as well as the hydrology of neighboring lakes or rivers.
Yangtze River is the largest river in China (6300 km); it is the fifth largest river in the world in terms of fresh water discharge (9.8 × 1011
/year), and the fourth largest in solid discharge (4.86 × 108
]. The Three Gorges Dam (TGD) is on the lower section of the upper reaches of the Yangtze River, and is the largest hydroelectric project in the world. The construction of the TGD began in 1993, and was completed in 2009, with some exceptions. The project has played a significant role in controlling frequent catastrophic floods downstream, generating hydropower (18,200 MW), water storage (12.4 billion m3
, 2003), and improving navigation at the upper reaches of the Yangtze River. However, the TGD affects ecosystems, known as TGD’s upstream and downstream effects, which stem from inundation, flow manipulation, and fragmentation [5
]. Dongting Lake is located in the middle and lower reaches of the Yangtze River. It is one of the largest freshwater lakes in China, and plays a significant role in regulating the amount of water entering the Yangtze River. The inflow from the Yangtze River carries an average of 4 million MT of sediment load per year [6
]. In recent years, the impoundment of the TGD has been implemented at the end of September. Before impoundment of the TGD, the water level in Dongting Lake was high and the lake was in its high water period, with the average water level reaching 27.36 m. After impoundment of the TGD, the water input from Yangtze River decreased and the lake entered its low water period with the average water level of 21.51 m. The phenomenon of dry–rewet cycles occurred and the sediments at some positions exposed from the water when the lake was in low water period.
As a distinct realm in aquatic ecosystems, sediments have higher biomass and microbe taxon richness than the bodies of water [7
]. Prokaryotes, especially bacteria, play a dominant role in fresh water lake sediments, promoting nutrient recycling and decomposition of organic or inorganic compounds [8
]. Therefore, a shift in bacterial communities may be one of the most sensitive indicators of environmental changes in the lake [10
]. The impoundment of the TGD has a significant effect on the Yangtze River, and it also affects the sediment in Dongting Lake because of its location. In recent years, the sediment in Dongting Lake has been influenced by the operation of TGD, such as the sediment deposition, the unsaturated sediment carrying capacity in the downstream-linked riverway, and riverbed sediment erosions occurring downstream after the impoundment of TGD, thereby causing siltation [11
]. In addition, microbial community changes in sediment also occur due to the impoundment of TGD. A few studies have addressed the microbial community composition affected by the TGD along the Yangtze River using more course-level culture-independent methods such as denaturing gradient gel electrophoresis (DGGE) and clone libraries [14
]. As one of the most significant microbes, bacteria in the sediment in Dongting Lake are crucial to the entire lake’s ecosystem.
The relationship between the Dongting Lake and the TGD has been studied by scholars from different perspectives, and most of the researchers focused on the early dry season in the downstream lakes, which is caused by TGD and nutrient characteristics, translocation, or sediment discharge [17
]. The potential effects of TGD impoundment on Dongting Lake have been investigated in previous studies that focused on prediction, verification, and analysis of soil properties and biomass in the early low-water period [18
]. However, few studies have been conducted on the effects of the impoundment of the TGD on the sediment bacterial community in Dongting Lake, and traditional techniques present inherent limitations in bacterial community studies. For example, when employing fingerprinting techniques, such as DGGE and phospholipid fatty acid analysis (PLFA), precise, accurate, and comprehensive description of microbial communities are difficult to obtain [21
]. Thus, the objective of the present study was to compare the sediment bacterial communities before and after impoundment of the TGD using Illumina Miseq. This study may provide new insights into the bacterial communities in the sediment of Dongting Lake.
2. Materials and Methods
2.1. Site Description and Sediment Sampling
Dongting Lake, which is the second largest freshwater lake in China, is located in Hunan Province (E 111°40’–113°10’, N 28°38’–29°45’). Dongting Lake is composed of a series of lakes and has three major lake districts, namely the eastern, southern, and western districts. The sample positions are shown in Figure 1
. Sediment 1 (S1) and Sediment 3 (S3) were from East Lake. Sediment 4 (S4) and Sediment 7 (S7) were from south lake. Sediment 8 (S8) was from west lake. Sediment 2 (S2) was from the outlet of Dongting Lake and the estuary of the Yangtze River. Sediment 5, 6, 9, and 10 (S5, S6, S9, and S10) were from the river estuary into Dongting Lake. Ten composite surface sediment samples (each N = 5
) were collected before (in July 2015, high water period,) and after (in October 2015, low water period) the impoundment of the Three Gorges Dam, and these samples were assigned as HS and LS, respectively. The weight of sample collected was sufficient for DNA extraction and analysis of physicochemical parameters. Sediment samples were put into sealed plastic bags and stored in a portable ice box, then transferred to the lab as quickly as possible and stored at −80 °C before analysis.
The sediment samples were divided into four groups. The samples of Group A (HS1, HS3, HS4, HS7 and HS8) and Group B (HS2, HS5, HS6, HS9 and HS10) were collected from the three lake districts of Dongting Lake and the river estuary into Dongting Lake during the high water period, respectively. The collection sites of the samples from Group C (LS1, LS3, LS4, LS7 and LS8) and Group D (LS2, LS5, LS6, LS9 and LS10) were the same as Groups A and B, respectively, and the samples were collected during the low water period.
2.2. Analysis of Physicochemical Parameters
The total phosphorus (TP) of the sediments was measured by using standardized methods and tests, and total nitrogen (TN) of the sediments was obtained as ammonium by colorimetry after the sample was digested with alkaline potassium persulfate (NaOH 0.24 mol·L−1
]. Organic matter (OM) content was calculated according to the loss on ignition to constant mass (4 h) at 550 °C [26
]. The pH of each sediment sample was measured in a 1:2.5 (w/v
) mixture of sediment with deionized water [27
]. The main properties of the collected sediments are shown in Table 1
2.3. DNA Extraction
Sediment samples were stored at −80 °C until DNA extraction. DNA was extracted from 1 g fresh sediment samples by using a PowerSoil DNA Isolation Kit (Mobio Laboratories Inc., San Diego, CA, USA), following manufacturer protocol. DNA concentration and purity were checked by running the samples on 1.2% agarose gels. All the extracted total DNA samples were stored in −80 °C before further analysis.
2.4. PCR Amplification of 16S rRNA Genes and Sequencing
The V4-V5 regions of bacterial 16S rRNA genes were amplified using the universal primers 515F (GTGCCAGCMGCCGCGGTAA) and 926R (CCGTCAATTCMTTTRAGTTT), these primers were chosen because of their high coverage of almost all phyla in conventional and metagenomic studies [28
]. The primers also contained the Illumina 5’ overhang adapter sequences for two-step amplicon library building, following manufacturer protocol for the overhang sequence. The amplification of bacteria gene fragments was achieved after two PCR steps.
PCR amplification was performed in a 25 μL reaction volume with 1 μL DNA template, 250 μM dNTPs, 0.25 μM of each primer, 1X reaction buffer, and 0.5 U Phusion DNA Polymerase (New England Biolabs, Ipswich, MA, USA). PCR cycling was carried out in a thermocycler under the following conditions: an initial denaturation at 94 °C for 2 min, followed by 25 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s and extension at 72 °C for 30 s, with a final extension of 72 °C for 5 min. The Illumina Nextera XT Index Kit (Illumina Inc., San Diego, CA, USA) with dual eight-base barcodes was used for multiplexing. Eight cycles of PCR reactions were used to incorporate two unique barcodes on both ends of the 16S amplicons. Cycling conditions consisted of one cycle at 94 °C for 3 min, followed by eight cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s. The final extension cycle was at 72 °C for 5 min.
Prior to library pooling, the barcoded PCR products were purified by using a DNA gel extraction kit (Axygen, Shanghai, China) and quantified by using the Qubit dsDNA HS Assay Kit (Life Technologies, New York, NY, USA). The libraries were sequenced by 2 × 300 bp paired-end sequencing on the MiSeq platform (Illumina, San Diego, CA, USA). Experiments were conducted at Tiny Gene Bio-Tech Co., Ltd. (Shanghai, China).
2.5. Statistical Analysis
The overlap between the forward and reverse reads was 180–190 bp approximately. The minimum quality score was 25 when merge the Illumina reads and the minimum overlap was 10 bp. The preprocessing of sequences was performed mainly by using MOTHUR 1.35.1 (University of Michigan, Ann Arbor, MI, USA) and by following the MiSeq analysis pipeline outlined in http://www.mothur.org/wiki/MiSeq_SOP
]. Operational taxonomic units (OTUs) are defined as groups among which sequence similarities were greater than 97%. The species richness estimators (the abundance-based coverage estimator ACE, Jackknife and Chao1), Shannon diversity index, and Simpson diversity index were calculated. In this study, data preprocessing and OTU-based analysis were performed by MOTHUR (University of Michigan, Ann Arbor, MI, USA). Canoco 4.5 (Microcomputer Power, Ithaca, NY, USA) was used with Monte Carlo permutation test to perform the redundancy analysis (RDA) based on population abundance and environment factors. LEfSe (University of Auckland, Auckland, New Zealand) was used to find indicator bacterial groups specific to the sediment samples [32
]. The statistical analysis was performed using SPSS 20.0 (International Business Machines Corporation, Armonk, NY, USA).
2.6. Accession Numbers
All of the sequencing data analyzed in the present study can be downloaded from the NCBI’s Sequence Read Archive using accession numbers SRR3354421 and SRR3354422.
The sediment bacterial community in downstream linked lakes was greatly influenced by the operation of large-scale water conservancy projects. In Dongting Lake, the bacterial communities had significant differences before and after impoundment of the TGD. The results show that the sediment samples before impoundment of the TGD had higher community diversity and richness than after impoundment. The most abundant phylum obtained by the sequence affiliated to the bacterial domain is Proteobacteria in both water periods. Betaproteobacteria and Deltaproteobacteria were highly abundant in the sediment samples before impoundment of the TGD. The abundance of Gammaproteobacteria in the sediment samples after impoundment of the TGD was high. Acidobacteria, Chloroflexi, Bacteroidetes, and Nitrospirae were also relatively abundant phyla in the sediment samples. The TP and DO concentrations affected bacterial communities of the samples from S1 and S9. The bacterial communities of the samples from S2 and S7 were related to the TN, ORP concentrations, and the water temperature.
The sediment bacterial community in downstream linked lakes is important to the entire lake’s ecosystem. Understanding the profiling of sediment bacterial community in downstream linked lakes before and after impoundment of the water conservancy projects is crucial to lake preservation and control. Taking a large downstream linked lake, Dongting Lake, as the example, new insights into the bacterial communities in the sediments of Dongting Lake and valuable references for such communities before and after impoundment of the TGD are provided.