Vertical diversity of sediment bacterial communities in two different trophic states of the eutrophic Lake Taihu, China

Vertical diversity of sediment bacterial communities in two different trophic states of the eutrophic Lake Taihu, China

Available online at www.sciencedirect.com JOURNAL OF ENVIRONMENTAL SCIENCES ISSN 1001-0742 CN 11-2629/X Journal of Environmental Sciences 2013, 25(6...

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Available online at www.sciencedirect.com

JOURNAL OF ENVIRONMENTAL SCIENCES ISSN 1001-0742 CN 11-2629/X

Journal of Environmental Sciences 2013, 25(6) 1186–1194

www.jesc.ac.cn

Vertical diversity of sediment bacterial communities in two different trophic states of the eutrophic Lake Taihu, China Keqiang Shao1 , Guang Gao1, ∗, Yongping Wang2 , Xiangming Tang1 , Boqiang Qin1 1. State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008, China. E-mail: [email protected] 2. Nanjing Institute of Hydraulic Research, Nanjing 210029, China Received 18 July 2012; revised 16 October 2012; accepted 18 December 2012

Abstract Vertical diversity of sediment bacterial communities in 2 different trophic states (macrophyte-dominated and algae-dominated) of the large shallow eutrophic Lake Taihu, China, were investigated using denaturing gradient gel electrophoresis (DGGE) and 16S rRNA sequence analysis. Clustering analysis of DGGE profiles showed that different clusters were recognized in different depths of sediment cores in the 2 lake trophic states. Analyses of the bacterial diversity, as estimated by the Shannon index (H ′ ), showed that different sediment layers of the macrophyte-dominated state had higher diversity than the algae-dominated state. In addition, bacterial diversity of the sediment in the macrophyte-dominated state changed abruptly throughout the layers, but bacterial diversity of the algae-dominated state decreased gradually with sediment depth. Phylogenetic analysis showed that Proteobacteria was the most abundant phylum in the middle sediment of the 2 lake trophic states. In the macrophyte-dominated state, clone sequences related to Betaproteobacteria (50.0%) were the most abundant, followed by Epsilonproteobacteria (21.1%), Acidobacteria (7.9%), Deltaproteobacteria (7.9%), Chloroflexi (7.9%), and Bacteroidetes (5.3%); whereas in the algae-dominated state, sequences affiliated with Betaproteobacteria (84.4%) were predominant, followed by Deltaproteobacteria (12.5%) and Acidobacteria (3.1%). Canonical correspondence analysis showed that organic matter and pH play key roles in driving the vertical changes of bacterial community composition. Key words: bacterial diversity; canonical correspondence analysis (CCA); denaturing gradient gel electrophoresis (DGGE); sediment core; macrophyte- and algae-dominated states; 16S rRNA DOI: 10.1016/S1001-0742(12)60122-3

Introduction Sedimentation is a process in which the interaction between deposition of organic material from the overlying water column and decomposition leads to the formation of a sediment layer (Ambrosetti et al., 2003). Sediment biogeochemical properties provide niches for metabolically diverse microorganisms (Zeng et al., 2008). Bacterial communities are important for biochemical cycling of the primary elements in freshwater lake sediments. Changes in environmental conditions, such as organic matter (OM) (Parkes et al., 2005) and nutrient availability (Nelson et al., 2007), can influence the bacterial community structure in the sediment. A previous study revealed that high levels of OM in sediment greatly stimulate prokaryotic activity and result in highly diverse prokaryotic populations (Parkes et al., 2005); therefore, investigation of vertical changes of bacterial communities in the sediment can elucidate * Corresponding author. E-mail: [email protected]

the benthic ecosystem processes in a eutrophic freshwater environment (Qu et al., 2008). Shallow lakes usually lack stratification and are known to have 2 alternative stable states (Scheffer et al., 1993). The macrophyte-dominated state is characterized by the presence of submersed macrophytes, clear water, and relatively low phytoplankton concentrations, whereas the algae-dominated state is characterized by the dominance of phytoplankton, frequent wind-driven resuspension of sediments, and high water turbidity (Qin et al., 2007; Wu et al., 2007). The bacterial community composition (BCC) of the water column was previously studied in 2 different ecological states (Haukka et al., 2006; Van der Gucht et al., 2001), as was the planktonic food web structure (Jeppesen et al., 1997). In Lake Taihu, the BCC of the water column and surface sediment were previously investigated in the macrophyte- and algae-dominated states (Wu et al., 2007; Shao et al., 2011). However, there tends to be homogeneity in the water due to its fluidity, while

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Vertical diversity of sediment bacterial communities in two different trophic states of the eutrophic Lake Taihu, China

sediments are unevenly distributed; thus, the distribution pattern of the bacterial community in the water column and sediment cores may not be the same. But, no study has attempted to describe the 2 states in terms of vertical changes of bacterial community structure in the sediment in this aquatic ecosystem. Lake Taihu is the third largest shallow eutrophic lake in China. According to spatial differences in physicochemical conditions and the plankton community structure, the lake can be divided into several ecological states. The aim of this study was to investigate the vertical changes of bacterial communities in the sediment of the macrophyte- and algae-dominated states within Lake Taihu. Additionally, the influences of several environmental factors on bacterial community distribution were assessed by multivariate analysis.

1 Materials and methods 1.1 Sampling sites Lake Taihu is a large, shallow, eutrophic lake located in the southeastern part of the delta of the Yangtze River. Xukou Bay is located in the eastern part of the lake and is typically a macrophyte-dominated state. Meiliang Bay, located in the northern part of Lake Taihu, is one of the most eutrophic areas and is typically an algae-dominated state (Qin et al., 2007). We worked at 2 sampling stations in the lake: SX (31◦ 5′ 43.5′′ N, 120◦ 19′ 48.7′′ E) in the macrophyte-dominated Xukou Bay and SM (31◦ 20′ 58.1′′ N, 120◦ 08′ 18.0′′ E) in the mouth of algae-dominated Meiliang Bay (Fig. 1). 1.2 Sample collection and chemical analyses Triplicate sediment cores located 0.1–0.5 km apart were collected with a piston corer from each sampling sites on Wuxi City 31°35′N

31°25′N

SM

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15 August 2009. Immediately after retrieval, they were sectioned into horizons of 12 depth strata with a sterile spatula (0–0.5, 0.5–1, 1–1.5, 1.5–2, 2–2.5, 2.5–3, 3–4, 4–5, 5–6, 6–8, 8–10, and 10–12 cm) using a slicing disc. The 72 sediment samples were transferred into sterile plastic containers, and stored frozen at –80°C. The pH was measured with specific electrodes (REX, PHB-5, INESA Scientific Instrument Co., Ltd., China). The sediment samples were dried with a laboratory freeze dryer (Alpha 1-2 LD, Martin Christ Instrument Co., Germany). Total nitrogen (TN), total phosphorus (TP) and OM were analyzed according to standard methods (Jin and Tu, 1990). 1.3 DNA extraction and PCR-DGGE Total genomic DNA was extracted using proteinase K, sodium dodecylsulfate, and cetyltrimethylammonium bromide concomitant with chloroform extraction and isopropanol precipitation as described by Zhou et al. (1996). Partial 16S rRNA gene fragments were PCR amplified using the bacterium specific primer F341 (5′ CCTACGGGAGGCAGCAG-3′ ) with a 40 bp GC-clamp attached to its 5′ end and the universal primer R907 (5′ -CCGTCAATTCMTTTGAGTTT-3′ ) (Muyzer et al., 1993). DNA samples extracted from 3 replicate layer sediments core were polymerase chain reaction (PCR) amplified respectively. After amplification, denaturing gradient gel electrophoresis (DGGE) was performed for each replicate sediment core, and we found that DGGE profiles of the three replicates are generally the same. Finally, the PCR products of 3 replicate sediments from each layer were pooled. The mixed PCR products for 24 sediment samples were loaded onto 8% (W/V) polyacrylamide gels cast in 1× TAE buffer (40 mmol/L Tris, 20 mmol/L acetic acid, and 1 mmol/L EDTA, pH 8.0), and made with denaturing gradients ranging from 45% to 65%. The DGGE was performed with a DGGE-2001 system (CBS Scientific Co., USA). The electrophoresis was conducted in 1× TAE buffer at 60°C initially at 20 V for 15 min and then at 100 V for 16 hr. After electrophoresis, the gels were stained with SYBR Green I solution (1:10000, Amresco, Inc., USA) for 30 min and photographed with a gel image analyzing system (Omega 10TM , Ultra-Lum Inc., USA). 1.4 Analysis of DGGE banding patterns

31°15′N

Lake Taihu

SX

31°05′N N 30°55′N

119°55′E 120°05′E 120°15′E 120°25′E 120°35′E Fig. 1 Map of Lake Taihu (China) with the sampling sites of the macrophyte-dominated Xukou Bay (SX) and mouth of algae-dominated Meiliang Bay (SM).

To assess the bacterial community in the different layers of the sediments, the DGGE data were used to generate a matrix of relational band intensities. A binary matrix according to the presence (1) or absence (0) of bands was determined with a gel documentation system, GelCompar II software (Applied Maths, USA). Cluster analysis of the DGGE banding patterns obtained from different layers sediment samples was performed with an unweighted pairwise grouping method with mathematical averages (UPGMA). Analysis of similarity (ANOSIM) was used to statistically test the differences in the profile of BCC

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using the Primer 6 package (Primer-E Ltd., UK) with the Bray-Curtis similarity indices. The ANOSIM generates a test statistic, R, and the magnitude of R is indicative of the degree of separation between groups, with a score of 1 indicating complete separation and 0 indicating no separation. As an estimate for the genetic diversity of the bacterial community, the Shannon index (H ′ ) was calculated using PAST (Paleontological Statistics v1.81) (Hammer et al., 2001). 1.5 Construction of clone library Two 16S rRNA gene clone libraries were constructed with sequences amplified from mixed bacterial 16S DNA templates retrieved from triplicate middle layer (5–6 cm) sediment samples: SX-7 from the macrophytedominated state and SM-7 from the algae-dominated state. Bacterial universal primers (forward primer 27F (5′ AGAGTTTGATCMTGGCTCAG-3′ ) and reversed primer 1492R (5′ -GGTTACCTTGTTACGACTT-3′ )) were used to amplify an approximately 1500 bp fragment of bacterial 16S rRNA genes (Newton et al., 2006). PCRs were carried out in a 50 µL mixture containing DNA templates, 5 µL of 10 × PCR buffer, 4 µL of dNTPs (2.5 mmol/L each, TaKaRa, Japan), 1.5 µL of each primer (10 µmol/L), and 0.5 µL of rTaq enzyme (5 U/µL) (TaKaRa, Japan). The PCR cycling was performed at 95°C for 5 min, 30 cycles at 94°C for 30 sec, 52 and 72°C for 1 min, with an additional 10 min of final extension at 72°C. For each sample, 3 replicate PCR reactions were carried out and pooled. The pooled products were purified immediately with the E.Z.N.A.® Cycle-Pure Kit (Omega, USA) and finally cloned into pMD18-T vector (TaKaRa, Japan) by using the protocol of the manufacturer. Ligated vectors were transformed into Escherichia coli DH5α chemically competent cells. The transformant clones were selected by a blue-white screening. The randomly chosen clones were checked for correct-length insertions by PCR using the vector primers RV-M and M13-47. 1.6 Sequencing and phylogenetic analysis Positive clones were randomly selected from two clone libraries sequenced using primer 27F (5′ AGAGTTTGATCMTGGCTCAG-3′ ), which was carried out at GenScript USA, Inc. (Nanjing, China). Sequences with 97% sequence similarity to any other were treated as a single phylotype. The obtained sequences were checked for potential chimeric sequences using the CHECK CHIMERA program (Maidak et al., 2000). Sequences containing no chimera were compared to the 16S rRNA sequences of the closest relative from the GenBank and Ribosomal Database Project databases to obtain a preliminary phylogenetic affiliation of the sequences. The phylogenetic trees were constructed by using the Molecular Evolutionary Genetics Analysis (MEGA) software package version 4.0 (Tamura et al.,

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2007). The robustness of the phylogenetic tree topology was confirmed by maximum parsimony analysis with 1000 bootstrap replications. Evolutionary distances were calculated using the Jukes-Cantor method (Tamura et al., 2004), and the neighbor-joining algorithm was used to generate the initial tree. 1.7 Statistical analyses Canonical correspondence analysis (CCA) was used to examine the influence of environmental variables (TN, TP, OM, and pH) on the vertical distribution of bacterial communities in the sediment. All data were log(x+1) transformed except pH. The CCA was performed with the software CANOCO 4.5 (SCIENTIA Software) using the unimodal method because detrended correspondence analysis run on species variables indicated that the length of the first axis was > 4. The significance of the first ordination and canonical axes together was assessed in permutation tests with 499 unrestricted Monte Carlo permutations. 1.8 Nucleotide sequence accession numbers The partial 16S rRNA gene sequences obtained in this study were submitted to the GenBank database with accession numbers HM854302–HM854364.

2 Results 2.1 Vertical changes of sediment properties The depth-related distribution of sediment properties (TN, TP, OM, and pH) of the macrophyte- and algae-dominated states in Lake Taihu (Fig. 2) shows that TN and OM contents were higher in the sediments of the macrophytedominated state, while TP content was higher in sediments of the algae-dominated state. The vertical variation of OM content was relatively small in the sediment of the algaedominated state, and it decreased gradually with depth in the 2 lake states. The pH of sediment in the 2 lake states decreased generally with depth but was higher in the sediment of the macrophyte-dominated state than that of the algae-dominated state. 2.2 Cluster analysis of DGGE profiles The vertical distribution of the bacterial communities in the sediment samples was evaluated by DGGE analysis (Fig. 3). On average 31.6 and 28.3 DGGE bands were obtained from the 12 sediment layers from the macrophyte- and algae-dominated states, respectively. The vertical changes in Shannon index (Fig. 4), indicate that H ′ of the macrophyte-dominated and the algae-dominated states ranged from 2.735 to 3.741 and from 3.000 to 3.523, respectively. The H ′ from the macrophyte-dominated state was higher than that of the algae-dominated state. To measure more clearly how the bacterial communities change with sediment depth, the DGGE patterns of bacterial communities in the different sediment layers of the

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2.4

34 Macrophyte (SX) Algae (SM)

32

2.0 TN (mg/g dw)

TP (mg/g dw)

Macrophyte (SX) Algae (SM)

2.2

30 28 26 24

1.8 1.6 1.4 1.2

22 20

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6 8 Depth (cm)

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14

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6 8 Depth (cm)

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8.3 Macrophyte (SX) Algae (SM)

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8.1

50 pH

OM (mg/g dw)

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40

8.0

30 7.9

20 10

7.8 6 8 10 12 14 0 2 4 6 8 10 12 14 Depth (cm) Depth (cm) Fig. 2 Vertical profiles of total nitrogen (TN), total phosphorus (TP), organic matter (OM) and pH in the sediment of the macrophyte-dominated Xukou Bay and mouth of algae-dominated Meiliang Bay in Lake Taihu. 0

2

4

Macrophyte (SX) M SX-0.5 SX-1 SX-1.5 SX-2 SX-2.5 SX-3

Algae (SM) M

SX-4 SX-5 SX-6

SX-8 SX-10 SX-12 M

M SX-0.5 SX-1 SX-1.5 SX-2 SX-2.5 SX-3

M

SX-4 SX-5 SX-6

SX-8 SX-10 SX-12 M

Fig. 3 DGGE banding profiles of bacterial 16S rRNA gene fragments from different layer sediment samples in the macrophyte-dominated Xukou Bay and mouth of algae-dominated Meiliang Bay in Lake Taihu. “M” refers to the standard lanes. Lanes SM (SX)-0.5, SM (SX)-1, SM (SX)-1.5, SM (SX)-2, SM (SX)-2.5, SM (SX)-3, SM (SX)-4, SM (SX)-5, SM (SX)-6, SM (SX)-8, SM (SX)-10, SM (SX)-12 represent 0–0.5, 0.5–1, 1–1.5, 1.5–2, 2–2.5, 2.5–3, 3–4, 4–5, 5–6, 6–8, 8–10,10–12 cm layer sediment samples.

macrophyte- and algae-dominated states were calculated on the basis of the UPGMA (Fig. 5). The resulting dendrograms of the DGGE patterns of the 12 different

sediment layer samples from the macrophyte- and algaedominated states showed distinct main clusters (Fig. 5). In the macrophyte-dominated state, the first cluster (I) was

Journal of Environmental Sciences 2013, 25(6) 1186–1194 / Keqiang Shao et al.

3.8

3.4 3.2 3.0 2.8

Macrophyte (SX) Algae (SM) 4

6 8 10 12 14 Depth (cm) Fig. 4 Vertical changes of Shannon-Wiener index of diversity (H ′ ) based on the number and relative intensities of the bands identified by DGGE analysis of different layer sediment samples of the macrophyte-dominated Xukou Bay and mouth of algae-dominated Meiliang Bay in Lake Taihu.

composed of the layers from 0.5 to 1.5 cm, the second one (cluster II) ranged from 1.5 to 3 cm, and the third one (cluster III) contained layers from 5 to 12 cm; the 3–4 cm sample was dissimilar to all other samples analyzed in the macrophyte-dominated state. In the algae-dominated state, the first cluster (I) was composed of the layers from 0.5 to 1.5 cm, the second one (cluster II) ranged from 1.5 to 3 cm, the third one (cluster III) from 3 to 6 cm, and the fourth cluster (IV) from 6 to 12 cm. The values of ANOSIM statistics R were 0.443 (p = 0.00825, peak area data comparison) and 0.428 (p = 0.00728, presence-absence data comparison), respectively, which demonstrated that there were significant variations in vertical distribution of bacterial community structure of the sediment between the macrophyte- and algae-dominated states (p < 0.01). 2.3 Phylogenetic composition of bacterial communities The frequencies of the cloned sequences obtained in the clone libraries from the middle sediment of the 2 lake states and their phylogenetic affiliation were summarized

2.4 Bacterial community composition in relation to sediment properties The CCA biplot consisting of DGGE data in relation to environmental variables (Fig. 7) shows that OM and pH contributed significantly to vertical changes in the BCC in the sediment (p < 0.05). The 2 axes explained 39.2% of the observed change in BCC. The different DGGE samples clearly clustered according to sampling site (Fig. 7), which also showed variations in the BCC between the macrophyte- and algae-dominated states in Lake Taihu.

3 Discussion The aim of this study was to investigate the vertical changes in bacterial community structure of the sediment in 2 different trophic states of Lake Taihu and to discuss the possible factors regulating their distribution and diversity. Previous studies described variations in bacterial community composition of the water column between 2 lakes with the 2 different ecological states (Van der Gucht et al., 2001); in lake mesocosms set up in 2 distinct ecological areas in shallow lakes in 2 different years (Haukka et al.,

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2.5-3 cm 2-2.5 cm Ⅱ 1.5-2 cm 0.5-1 cm 0-0.5 cm Ⅰ 1-1.5 cm 5-6 cm 4-5 cm Ⅲ 8-10 cm 6-8 cm 10-12 cm 3-4 cm

92

Algae (SM)-similarity (%)

Macrophyte (SX)-similarity (%)

90

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84

Diversity index (H′)

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(Table 1). The phylotypes that comprise the bacterial community of the clone libraries from the middle sediments in the 2 lake states (Fig. 6) indicate that the bacterial species composition of the 2 clone libraries differed substantially at the phylum levels. In the macrophyte-dominated state library of Lake Taihu, the bacterial community of the middle sediment was dominated by Betaproteobacteria (50.0%), Epsiloproteobacteria (21.1%), Acidobacteria (7.9%), Deltaproteobacteria (7.9%), Chloroflexi (7.9%), and Bacteroidetes (5.3%). In contrast, in the algae-dominated state library of the lake, the bacterial community of the middle sediment was dominated by Betaproteobacteria (84.4%), Deltaproteobacteria (12.5%), and Acidobacteria (3.1%).

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1-1.5 cm 0-0.5 cm 0.5-1 cm 2.5-3 cm 2-2.5 cm 1.5-2 cm 10-12 cm 8-10 cm 6-8 cm 5-6 cm 4-5 cm 3-4 cm









Fig. 5 Dendrogams of the bacterial communities in the different layers sediments of the macrophyte-dominated Xukou Bay and mouth of algaedominated Meiliang Bay in Lake Taihu. Similarities were calculated using the unweighted-pair group method.

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sp

sp

Spirochaeiales Spirochaeiales Spirochaeiales

Fig. 6 Phylogenetic tree for the 16S rRNA gene sequences obtained from two clone libraries of the middle layer sediment samples from the macrophytedominated Xukou Bay (TH-c2) and mouth of algae-dominated Meiliang Bay (TH-z2) in Lake Taihu. Numbers of clones are given in parentheses. The neighbor-joining algorithm was used to construct the initial tree in MEGA4. Bootstrap analysis was conducted using 1000 replicates. Bootstrap values above 50% for branches are shown. The evolutionary distances were computed using the Jukes-Cantor method and expressed as number of base substitutions per site.

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Table 1 Phylogenetic affiliation of clones contained in the two environmental 16S rRNA libraries constructed with middle sediment samples from the algae-dominated state (mouth of Meiliang Bay) and the macrophyte-dominated state (Xukou Bay) within Lake Taihu in August 2009 Phylogenetic group

Surface sediment∗ No. of clones Frequencies (%) Macrophyte Algae Macrophyte Algae

Middle sediment No. of clones Frequencies (%) Macrophyte Algae Macrophyte Algae

Alphaproteobacteria Betaproteobacteria Deltaproteobacteria Gammaproteobacteria Cyanobacteria Epsilonproteobacteria Acidobacteria OP10 Planctomycetes Bacteroidetes Verrucomicrobia Chloroflexi Unidentified Total

0 3 10 4 0

1 10 9 5 4

0 8.3 27.8 11.1 0

2.3 23.3 20.9 11.6 9.3

5 1 0 4 5 1 3 36

5 0 4 1 1 1 2 43

13.9 2.8 0 11.1 13.9 2.8 8.3

11.6 0 9.3 2.3 2.3 2.3 4.7

0 19 3 0 0 8 3 0 0 2 0 3 0 38

0 50.0 7.9 0 0 21.1 7.9 0 0 5.3 0 7.9 0

0 84.4 12.5 0 0 0 3.1 0 0 0 0 0 0

Data are cited from Shao et al., 2011.

1.0



0 27 4 0 0 0 1 0 0 0 0 0 0 32

SX-0.5

SM-3 *pH

SX-3

Axis 2 (5%)

SX-1 * OM

SM-2

SX-4

TN

SX-2.5

SX-1.5 SX-2

TP

SM-2.5 SM-4

SX-8

SM-5 SM-6

SX-10 SM-8 SM-10 SM-0.5 SM-12 SM-1

SX-5 -0.6

SX-6 SX-12

SM-1.5

-1.0

0.6 Axis 1 (17.1%) Fig. 7 Canonical correspondence analysis biplots show denaturing gradient gel electrophoresis (DGGE) data relationships to four environmental factors. Environmental variables marked with asterisks were significant (p < 0.05).

2006); between 2 different ecological states of the shallow Lake Taihu (Wu et al., 2007; Tang et al., 2010); and in the surface sediment between the 2 different ecological states of Lake Taihu (Shao et al., 2011). Our investigation covers the sediment column of the 2 different ecological states in Lake Taihu. The DGGE results showed that H ′ from the sediment of the macrophyte-dominated state did change abruptly throughout the layers, and H ′ from the algae-dominated state decreased gradually with sediment depth. The differences in vertical distribution of bacterial diversity between the macrophyte-dominated state (Xukou Bay) and algae-dominated state (Meiliang Bay) could partially be

explained by the different environmental conditions of the 2 lake states. In Lake Taihu, Meiliang Bay was the most eutrophic and macrophyte-absent region, which is characterized by the dominance of phytoplankton (Qin et al., 2007; Wu et al., 2007). Moreover, it receives most of the inflow (Qin et al., 2004) as well as the import of large amounts of soil particles from the watershed due to intensive agriculture and soil erosion (Dokulil et al., 2000; Qin et al., 2004). In contrast, Xukou Bay was characterized by submersed macrophytes, clear water, and diverse communities of invertebrates (Qin et al., 2007; Wu et al., 2007). Another factor that contributes to differences between the macrophyte-dominated state and algae-dominated state is the presence or absence of submersed macrophytes. The macrophyte roots may affect the vertical distribution of nutrients in the sediment, resulting in changes in the vertical distribution of bacterial diversity in the sediment. A previous study also demonstrated that submersed macrophytes may exert direct and indirect influences on the abundance and composition of planktonic bacterial communities (Wu et al., 2007). Our results clearly indicate significant variations in BCC of the sediment column between the macrophyte- and algae-dominated states, possibly due to significant variations in the organic fractions of the sediment between the 2 states. In the macrophyte-dominated state of Lake Taihu, the organic fractions of the sediment mainly formed from decomposing and dead residues of large vascular plants. In contrast, the organic fractions of the sediment of the algae-dominated state mainly derived from the suspended particulate matter of urban sewage and the organic remains of algae (Qin et al., 2004). A previous report also showed a site-specific variation in BCC of the surface sediment between the 2 lake states (Shao et al., 2011). To reveal the exact vertical variation of the structure of the sediment bacterial community between the 2 lake states, however, more detailed in situ studies are needed at other different

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locations of the 2 lake states. We also found the interesting phenomenon that the 3–4 cm sediment sample is far from the other clusters in the macrophyte-dominated state and has the lowest number of DGGE bands (Fig. 5), possibly related to the nutrient level of this sediment layer. We can find the emergence of a point of inflexion in the 3–4 cm layer sediment sample from the vertical profile of sediment properties (Fig. 2). The concentrations of TN, TP, and OM changed abruptly within the upper 4 cm then decreased gradually in the sediment of the macrophyte-dominated region. In addition, this may be caused by biases introduced into our results associated with many research procedures such as sample collection, DNA extraction, PCR amplification (Kanagawa, 2003; Zeidner and Beja, 2004), and other subsequent operations. The phylogenetic composition of the bacterial community of the surface sediment in the macrophyte- and algae-dominated states of Lake Taihu has been previously investigated (Shao et al., 2011). When comparing bacterial diversity between the surface sediment and the middle sediment in the same typical ecological areas of Lake Taihu, we are now able to show that the surface sediment samples had significantly greater bacterial diversity than the middle sediment samples in the 2 lake states (Table 1). A plausible explanation for the results is that the surface sediments contain higher concentrations of nutrients and resources used by bacteria than the middle sediment, an interpretation consistent with the higher percentages of OM in surface sediments than middle sediments (Fig. 2). OM from the middle sediments is likely more recalcitrant than OM from the surface sediment (Ishiwatari, 1985). More abundant and labile OM may support elevated bacterial diversity and richness through increased niche partitioning (Dykhuizen, 1998). In our study, we found that the bacterial groups detected by sequence analysis are commonly found in freshwater environments (Haukka et al., 2006). Proteobacteria were the most abundant bacterial groups in the two 16S rRNA clone libraries constructed with sediment samples from the macrophyte- and algae-dominated states. This finding is consistent with several previous studies demonstrating that Proteobacteria are the dominant group in most freshwater sediments (Bowman and McCuaig, 2003; Wilms et al., 2006). Most Proteobacteria are known to easily metabolize degradable organic substrates (Wilms et al., 2006); therefore, their dominance of the sediment of this eutrophic lake is not unexpected. The existence of Bacteroidetes in the sediment is related to their physiological characteristics. Surface-dependent gliding motility is an important and widespread characteristic of these bacteria. Second, Bacteroidetes can efficiently degrade a variety of high molecular weight compounds (Kirchman, 2002). Previous studies have demonstrated that bacterioplankton belonging to the Bacteroidetes is abundant in lakes associated with

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cyanobacterial blooms (Eiler and Bertilsson, 2004; Tang et al., 2010). Acidobacteria are a newly devised phylum of Bacteria, whose members have only recently been discovered and the large majority of which have not been cultured; the ecology and metabolism of these bacteria is not well understood (Quaiser et al., 2003). However, these bacteria may be an important contributor to ecosystems, since they are particularly abundant within soils (Eichorst et al., 2007). Our CCA results showed that OM and pH significantly influenced the bacterial community structure in the sediment, in agreement with previous studies. In Lake Xuanwu, China, OM was identified as a driving force of changes in the bacterial community in the sediments (Zeng et al., 2009). In a mercury-polluted lake, OM has previously been reported to have a significant effect on the bacterial community composition in the sediment (Macalady et al., 2000). The correlation between pH and bacterial community structure has also been reported elsewhere. The pH may merely reflect changes in other environmental factors, such as the availability of ions and trace metals, which can have both inhibitory and stimulative effects on the bacterial community (Koski-V¨ah¨al¨a et al., 2001). Furthermore, a fluctuating pH may also influence the bacterial community through direct biological mechanisms (Yannarell and Triplett, 2005). Acknowledgments This work was supported by the Key Program of Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences (No. NIGLAS2012135002), the National Key Natural Science Foundation of China (No. 41230744), the Open Foundation from the State Key Laboratory of Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences (No. 2010SKL008). We thank Dr. Guangwei Zhu from Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, for providing much appreciated field support for sample collections.

References Ambrosetti W, Barbanti L, Sala N, 2003. Residence time and physical processes in lakes. Journal of Limnology, 62(1): 1–15. Bowman J P, McCuaig R D, 2003. Biodiversity, community structural shifts, and biogeography of prokaryotes within Antarctic continental shelf sediment. Applied and Environmental Microbiology, 69(5): 2463–2483. Dokulil M T, Chen W, Cai Q, 2000. Anthropogenic impacts to large lakes in China: the Taihu example. Aquatic Ecosystem Health and Management, 3(1): 81–94. Eichorst S A, Breznak J A, Schmidt T M, 2007. Isolation and characterization of soil bacteria that define Terriglobus gen. nov., in the phylum Acidobacteria. Applied and Environmental Microbiology, 73(8): 2708–2717. Eiler A, Bertilsson S, 2004. Composition of freshwater bacterial communities associated with cyanobacterial blooms in four Swedish lakes. Environmental Microbiology, 6(12): 1228–

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1243. Hammer Ø D, Harper A T, Ryan P D, 2001. PAST: Paleontological Statistics Software Package for education and data analysis. Palaeontologia Electronica, 4: 9. http://palaeoelectronica.org/2001 2001/past/issue2001 2001.htm. Haukka K, Kolmonen E, Hyder R, Hietala J, Vakkilainen K, Kairesalo T et al., 2006. Effect of nutrient loading on bacterioplankton community composition in Lake Mesocosms. Microbial Ecology, 51(2): 137–146. Ishiwatari R, 1985. Geochemistry of humic substances in lake sediments. In: Humic Substances in Soil, Sediment, and Water: Geochemistry, Isolation, and Characterization (Aiken G R, McKnight D M, eds.). Wiley, New York. 147–180. Jeppesen E, Sondergaard M, Christoffersen K, 1997. The Structuring Role of Submersed Macrophytes in Lakes. Springer, New York. Jin X C, Tu Q Y, 1990. Lake Eutrophication Survey Specification. Environmental Science Press, Beijing. 208–230. Kanagawa T, 2003. Bias and artifacts in multitemplate polymerase chain reactions (PCR). Journal of Bioscience and Bioengineering, 96(4): 317–323. Kirchman D L, 2002. The ecology of Cytophaga-Flavobacteria in aquatic environments. FEMS Microbiology Ecology, 39(2): 91– 100. Koski-V¨ah¨al¨a J, Hartikainen H, Tallberg P, 2001. Phosphorus mobilization from various sediment pools in response to increased pH and silicate concentration. Journal of Environmental Quality, 30(2): 546–552. Macalady J L, Mack E E, Nelson D C, Scow K M, 2000. Sediment microbial community structure and mercury methylation in mercury-polluted Clear Lake, California. Applied and Environmental Microbiology, 66(4): 1479–1488. Maidak B L, Cole J R, Lilburn T G, Parker C T Jr, Saxman P R, Stredwick J M et al., 2000. The RDP (Ribosomal Database Project) continues. Nucleic Acids Research, 28(1): 173–174. Muyzer G, DeWaal E C, Uitterlinden A G, 1993. Profiling of complex microbial populations by denaturing gradient del electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Applied and Environmental Microbiology, 59(3): 695–700. Nelson D M, Ohene-Adjei S, Hu F S, Cann I K O, Mackie R I, 2007. Bacterial diversity and distribution in the Holocene sediments of a northern temperate lake. Microbial Ecology, 54(2): 252– 263. Newton R J, Kent A D, Triplett E W, McMahon K D, 2006. Microbial community dynamics in a humic lake: differential persistence of common freshwater phylotypes. Environmental Microbiology, 8(6): 956–970. Parkes R J, Webster G, Cragg B A, Weightman A J, Newberry C J, Ferdelman T G et al., 2005. Deep sub-seafloor prokaryotes stimulated at interfaces over geological time. Nature, 436(7049): 390–394. Qin B Q, Hu W P, Chen W M, 2004. Process and Mechanism of Environmental Changes of Lake Taihu. Science Press, Beijing. Qin B Q, Xu P Z, Wu Q L, Luo L C, Zhang Y L, 2007. Environmental issues of Lake Taihu, China. Hydrobiologia, 581(1): 3–14. Qu J H, Yuan H L, Wang E T, Li C, Huang H Z, 2008. Bacterial diversity in sediments of the eutrophic Guanting Reservoir, China, estimated by analyses of 16S rDNA sequence. Biodiversity and Conservation, 17(7): 1667–1683.

Vol. 25

Quaiser A, Ochsenreiter T, Lanz C, Schuster S C, Treusch A H, Eck J et al., 2003. Acidobacteria form a coherent but highly diverse group within the bacterial domain: evidence from environmental genomics. Molecular Microbiology, 50(2): 563– 575. Scheffer M, Hosper S H, Meijer M L, Moss B, Jeppesen E, 1993. Alternative equilibria in shallow lakes. Trends in Ecology and Evolution, 8(8): 257–279. Shao K Q, Gao G, Qin B Q, Tang X M, Wang Y P, Chi K X et al., 2011. Comparing sediment bacterial communities in the macrophyte-dominated and algae-dominated areas of eutrophic Lake Taihu, China. Canadian Journal of Microbiology, 57(4): 263–272. Tamura K, Dudley J, Nei M, Kumar S, 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24(8): 1596–1599. Tamura K, Nei M, Kumar S, 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences of the United States of America, 101(30): 11030–11035. Tang X M, Gao G, Chao J Y, Wang X D, Zhu G W, Qin B Q, 2010. Dynamics of organic-aggregate-associated bacterial communities and related environmental factors in lake Taihu, a large eutrophic shallow lake in China. Limnology and Oceanography, 55(2): 469–480. Van der Gucht K, Sabbe K, De Meester L, Vloemans N, Zwart G, Gillis M et al., 2001. Contrasting bacterioplankton community composition and seasonal dynamics in two neighbouring hypertrophic freshwater lakes. Environmental Microbiology, 3(11): 680–690. Wilms R, K¨opke B, Sass H, Chang T S, Cypionka H, Engelen B, 2006. Deep biosphere-related bacteria within the subsurface of tidal flat sediments. Environmental Microbiology, 8(4): 709– 719. Wu Q L, Zwart G, Wu J F, Kamst-van Agterveld M P, Liu S J, Hahn M W, 2007. Submersed macrophytes play a key role in structuring bacterioplankton community composition in the large, shallow, subtropical Taihu Lake, China. Environmental Microbiology, 9(11): 2765–2774. Yannarell A C, Triplett E W, 2005. Geographic and environmental sources of variation in lake bacterial community composition. Applied and Environmental Microbiology, 71(1): 227–239. Zeidner G, Beja O, 2004. The use of DGGE analyses to explore eastern Mediterranean and Red Sea marine picophytoplankton assemblages. Environmental Microbiology, 6(5): 528–534. Zeng J, Yang L Y, Li J Y, Liang Y, Xiao L, Jiang L J et al., 2009. Vertical distribution of bacterial community structure in the sediments of two eutrophic lakes revealed by denaturing gradient gel electrophoresis (DGGE) and multivariate analysis techniques. World Journal of Microbiology and Biotechnology, 25(2): 225–233. Zeng J, Yang L Y, Liang Y, Li J Y, Xiao L, Jiang L J et al., 2008. Spatial distribution of bacterial communities in sediment of a eutrophic lake revealed by denaturing gradient gel electrophoresis (DGGE) and multivariate analysis. Canadian Journal of Microbiology, 54(12): 1053–1063. Zhou J Z, Bruns M A, Tiedje J M, 1996. DNA recovery from soils of diverse composition. Applied and Environmental Microbiology, 62(2): 316–322.