Temporal variation of coastal surface sediment bacterial communities along an environmental pollution gradient

Marine Environmental Research 70 (2010) 56e64

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Temporal variation of coastal surface sediment bacterial communities along an environmental pollution gradient V. Thiyagarajan b,1, M.M.Y. Tsoi a,1, W. Zhang a, P.Y. Qian a, * a b

Department of Biology/Coastal Marine Laboratory, The Hong Kong University of Science and Technology, Kowloon, Hong Kong SAR, Hong Kong The Swire Institute of Marine Science and School of Biological Sciences, The University of Hong Kong, Pok Fu Lam Road, Hong Kong SAR, Hong Kong

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 December 2008 Received in revised form 27 February 2010 Accepted 2 March 2010

Terminal restriction fragment length polymorphism analysis (T-RFLP) was used to track the changes of bacterial community compositions (BCC) in coastal surface sediments along an environmental pollution gradient between 2004 and 2006. BCC in the chronically contaminated sites showed the largest deviation from those in the adjacent sites. Surprisingly, BCC at two contrasting environments (oceanic vs. riverinfluenced) were more similar. Unexpectedly, the BCC did not recover (when compared to oceanic control site) even after 5 years of pollution abatement initiatives in Victoria Harbour, Hong Kong. On the other hand, disposal of treated sewage for 5 years in one of the sites did not significantly affect the BCC. A striking seasonal variation in the BCC was observed at only the polluted sites. Although factors other than pollution gradients may explain the observed BCC patterns, the information presented here can be useful in predicting long-term effects of pollution on BCC. Furthermore, this study suggests that BCC analysis using T-RFLP is a faster, reliable and easier approach to monitor microbenthic community response to environmental pollution gradient in coastal sediments. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Bacterial community Environmental impact Multiple stresses T-RFLP Sediment pollution Victoria harbour 16S-rDNA Hong Kong

1. Introduction Bacteria in marine sediments exhibit extensive spatial heterogeneity in terms of abundance, diversity, and activity in both horizontal and vertical dimensions (Urakawa et al., 1999; Zhang et al., 2008a,b). In addition, sediment bacterial communities are susceptible to anthropogenic disturbances such as changes in heavy metal contamination and organic enrichment. For example, accumulation of toxic components (DDT and heavy metals) in sediments can lead to a decrease in microbial diversity or inhibit microbial activity in sediments (Han et al., 1999; Gillan et al., 2005; Duran et al., 2008). Thus bacterial diversity and their spatiotemporal variation in coastal surface sediments are highly vulnerable to anthropogenic damage (Ogilvie and Grant, 2008). But the response of bacterial communities in coastal surface sediments to anthropogenic disturbances and environmental gradients has been rarely investigated (Bissett et al., 2006; Hewson et al., 2007). Molecular fingerprinting techniques have provided a powerful alternative to the culture-dependent techniques and allowed us to gain new insights into variation in bacterial diversity in response to * Corresponding author. Tel.: þ852 2358 7331; fax: þ852 2358 1559. E-mail address: [email protected] (P.Y. Qian). 1 These authors contributed equally to this work. 0141-1136/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2010.03.002

environmental heterogeneity. Among fingerprinting techniques, the terminal restriction fragment length polymorphism (T-RFLP) analysis is effective in discriminating between microbial communities in a variety of environments (Liu et al., 1997; Nakano et al., 2008). T-RFLP analysis is inexpensive and fast with the ability to screen several hundred samples a day (Zhang et al., 2008c). Thus, it is routinely used as a survey tool to monitor the dynamics of bacterial communities (water column: Wu et al., 2004; Zhang et al., 2008c; marine sediments: Yoza et al., 2007; biofilm: Qian et al., 2003) or to quickly identify samples of interest (Lau et al., 2005). Marine sedimentary bacteria have rapid growth rates and individual species within a community often show different responses to changing environments (Ford et al., 2005). Any shift in environmental condition or pollution loading in the ecosystem, may therefore directly affect the bacterial community composition, especially on surface sediments (Paerl et al., 2003). Changes in softbottom macrobenthic community composition have been widely used for detecting and monitoring the biological effects of human activities in the marine environment (e.g. Shin and Ellingsen, 2004). However, changes in the bacterial community composition in surface marine sediments have been mostly ignored in such an approach (Bissett et al., 2006). If the bacterial community composition in soft-benthic habitat is determined by their environment, then pollution loading or organic enrichment is expected to shift

V. Thiyagarajan et al. / Marine Environmental Research 70 (2010) 56e64

their composition, and a counter shift toward the original community should be evident after the cessation or reduction of pollution discharge (Yoza et al., 2007). The surface sediments in coastal Hong Kong waters provide ideal samples to test the response of bacterial communities to environmental pollution gradients (Zhang et al., 2008a,b) and their recovery to pollution abatement. Because of the uncontrolled disposal of domestic and industrial wastewater into the Victoria Harbour, Hong Kong, especially from the 1960s to the 1980s, it has become seriously contaminated (HKEPD 2004, 2006). The harbour, especially VHE and VH areas (see Fig. 1), received about 1.5 Mt of untreated sewage and industrial wastewater per day until recently. Since 2002, however, sewage effluents are no longer directly disposed into the central harbour and sewage is collected, treated, and then disposed into the harbour at VHW (see Fig. 1). In addition, as one of the busiest shipping ports in the world, the harbour also receives shippingrelated contaminants daily. As a result, sediments in the central harbour area (VH in Fig. 1) are heavily polluted by heavy metals, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, and a very high concentration of sulfide (HKEPD, 2006). The severity of sediment contamination decreases towards both the eastern (oceanic) and western (the Pearl River estuary) sides, forming a clear pollution gradient (Zhang et al., 2008b). The purpose of this study was to assess the spatio-temporal variability of the surface sediment bacterial communities along an environmental pollution gradient generated by the discharge of sewage and the Pearl River in sub-tropical shallow coastal waters. The following specific questions were addressed: 1) Are there any seasonal and/or inter-annual changes in site-specific bacterial community composition along the environmental pollution gradient in Victoria Harbour? and 2) With respect to species diversity, do sites close to the sewage discharge area support sediment bacterial communities distinct from those in control sites? To address these questions, we used the DNA fingerprinting technique T-RFLP to analyze surface sediment samples collected between 2004 and 2006 from five sites across a 5 km transect.

57

2. Materials and methods

adjacent sites, Hong Kong (Fig. 1). The hydrography in the study area varies at spatio-temporal scales (Shin and Ellingsen, 2004). As a result of freshwater outflow from the Pearl River in the western side of Victoria Harbour, an increasing trend in salinity concentration from west to east occurs during summer (May to September). In winter, the Kuroshio Current (high salinity and temperature) affects the hydrography in this region. On top of these natural events, human impacts through pollution and sewage discharge are serious in the study area (see Introduction). Therefore, this area was chosen for an investigation of spatio-temporal variations in softbottom biochemical characteristics (due to seasonal environmental variation and anthropogenic pollution) on bacterial community composition in surface sediments. Sediment samples from five sites were used in this study. These sites are hereafter referred to as TLC (Tung Lung Chau), VHE (Victoria Harbour east), VH (Victoria Harbour), VHW (Victoria Harbour west), and PC (Peng Chau) (Fig. 1). All these sites are located at relatively similar depths (7e15 m). The western site (PC) is heavily affected by the discharge from the Pearl River and the eastern site (TLC) is oceanic. The transition sites, VH, VHE and VHW, are polluted by domestic sewage (treated and/or untreated) discharges from about 3.5 million people (Wong et al., 2000). Samples were collected during six sampling dates: summer 2004 (August 13, 2004), winter 2004 (December 14, 2004), summer 2005 (July 17, 2005), winter 2005 (December 12, 2005), winter 2006 (December 10, 2006), and summer 2006 (June 21, 2006). On each sampling date, hydrographic and bacterioplankton data were collected but presented elsewhere (see Zhang et al., 2008c). To obtain surface sediments of similar grain size, extra care was taken in selecting the precise sampling locations. In each sampling, four homogenous (well-mixed before sampling) surface sediment samples (from top 1 cm) per site and sampling date were collected using a Gravity Core (KC DenMerk). Samples were transported back to the laboratory in dry ice and kept at 80  C until the extraction of total genomic DNA. A portion of sediment samples was frozen at 20  C for subsequent analysis of biochemical characteristics, while the chlorophyll a (Chl a) content was determined immediately after sample collection.

2.1. Site description and sediment sample collection

2.2. Biochemical analysis of sediments

The sediment samples for this study were collected along an environmental and pollution gradient in Victoria Harbour and

For both biochemical and bacterial community analysis, 120 samples (4 replicates  5 sites  6 sampling dates) were used. The

Fig. 1. A map showing the locations of sediment collection sites in Victoria Harbour, Hong Kong. TLC e Tung Lung Chau; VHE e Victoria Harbour east; VH e Victoria Harbour; VHW e Victoria Harbour west; PC e Peng Chau. The short arrow located close to VHW shows the treated sewage discharge point.

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physical properties of were determined in terms of their mean grain size and percent water content (Thiyagarajan et al., 2006). To estimate the organic matter content of each sample, total organic carbon (TOC), nitrogen (TN), and sulfur contents were determined for homogenized samples after acidification with 1 M hydrochloric acid (Hedges and Stern,1984) using a CHNS Perkin Elmer 2400 elemental analyzer. Chl a concentration was measured as a proxy of microalgal biomass. Acid volatile sulfide, organic and inorganic pollutant levels were also measured in those sediment samples (see Zhang et al., 2008a,b for details). Briefly, those pollution monitoring results showed that both TLC and PC had relatively lower concentration of trace metals compared to VH and VHW (Table 1). A similar trend was also found in the case of major elements.

ethanol precipitation and dissolved in 20 ml of sterile dd H2O. Ten ml of purified products together with 0.5 ml of internal standard (ET900R, Amersham) were denatured at 95  C for 2 min, snap cooled on ice, and subjected to electrophoresis on a MegaBACE genetic analyzer (Amersham) operated in the genotyping mode. After electrophoresis, the basic T-RF calling was performed using a Genetic Profiler in the MEGABACE software package (Amersham). For each sample, peaks over a threshold of 50 fluorescence units were used. To avoid detection of primers and uncertainties in the determination of fragment size, T-RFs of <30 bp and >900 bp were excluded from the data analysis (Zhang et al., 2008c).

2.3. DNA extraction

Bacterial community data (T-RFLP profiles) were analyzed using multivariate techniques. Formal significance tests for differences among sampling dates and sites were performed using the crossed two-way ANOSIM (analysis of similarities) randomization/permutation test (Clarke and Warwick, 1994) from the similarity matrix based on Dice coefficient. The similarity result was calculated based on the total number of T-RFs observed in all samples and the presence or absence of these T-RFs in individual samples (Zhang et al., 2008c). Multidimensional scaling (MDS) ordination was subsequently used to map the spatio-temporal relationships in the similarities for all five sites and six sampling dates. Pairwise comparisons of bacterial community composition between any pair of sites in each sampling date were determined using ANOSIM randomization permutation test with 5000 permutations. In order to verify how much of the variability in the T-RFLP bacterial community composition profile/data matrix was explained by the observed sediment characteristics, the “DISTLM forward” computer program was utilized (Anderson, 2003). In the marginal test, effects of each sediment characteristics on T-RFLP profiles were analyzed separately. Sediment variables were then subjected to forward selection procedure using sequential R2 selection criterion. In sequential tests, the amount of variance explained by each sediment characteristics added to the model is conditional on the variable already in the model. The P-values were obtained using 9999 permutations of the normalized and logtransformed data. All tests were based on BrayeCurtis dissimilarities. Following DISTLM, ordination method of redundancy analysis (RDA) was used to visualize the relationship between T-RFLP data and sediment characteristics. The computer package PRIMER (Clark and Gorley, 2001) with PERMANOVA was employed for all nonmetric ordinations.

Total DNA was extracted from the sediment samples according to Forthin et al. (2004) with some modifications. Approximately 0.4 g of sediment samples were transferred into 2 ml screw cap microcentrifuge tubes together with 0.1 g of glass beads and washed sequentially in three washing solutions followed by vortex mixing and centrifugation. The supernatant was discarded and the washed sediment sample was homogenized in 0.4 ml of extraction buffer using a mini-beater (BioSpec Products, Bartlesville, OK, USA). The cells were lysed by using three freezeethaw cycles. The total DNA was extracted twice with an equal volume of chloroformeisooamlyalcohol (24:1 vol/vol). The aqueous phase was collected and the DNA was precipitated and washed using isopropanol and then 70% ethanol. The extracted DNA was dissolved in 100 ml sterile dd H2O and frozen at 20  C until further analysis. 2.4. PCR amplification of the 16S rRNA gene PCR of the bacterial 16S rRNA gene (rDNA) in the crude DNA extract was performed in a total volume of 25 ml containing 0.1e0.2 mg of DNA template, 200 mM of each of deoxynucleoside triphosphate, 0.75 U of DNA Taq polymerase (Takara Biotechnology, Japan) and 0.2 mM of each of the domain specific primers: 8F (labeled at the 5’end with a 6-carboxy fluorescen (FAM) dye) and 1055R (Edwards et al., 1989; Reiter et al., 2002). PCR was performed in the following thermal cycles: 95  C for 2 min; 10 touchdown cycles of 95  C for 1 min, 60  C (reduced to 51  C in increments of 1  C cycle1) for 1 min and 72  C for 1 min; additional 15 cycles with a constant annealing temperature of 45  C; and 72  C for 5 min. Successful amplification of DNA was verified by electrophoresis of 3.5 ml of PCR products in 1.5% of agarose gel in TAE buffer (40 mM Triseacetate, 1 mM EDTA, pH 8.0).

2.6. Statistical analysis

3. Results 3.1. Spatio-temporal variation in sediment biochemical properties

2.5. T-RFLP analysis of bacterial communities PCR products were cleaved with 10 U of the restriction enzyme Hae III at 37  C for 6 h. Cleaved PCR products were purified by

All sediment samples used in this study were mostly muddy (mean grain size ranged from 45 mm to 65 mm) with 50e65% of water content with no significant difference among the five sites

Table 1 Mean (S.D. of three replicates per site and each site was sampled six times during 2004e2006) concentrations (mg/kg, dry weight) of trace metals and major elements; and simultaneously extracted metal (SSEM) and acid volatile sulfide (AVS) values (in %) in the study sites. Data from six sampling times were pooled. See Fig. 1 for site abbreviations. Sites

Trace metal concentration Cu

TLC VHE VH VHW PC

12.5  88.4  127.6  58.1  21.9 

2.3 13.34 36.7 6.5 1.8

16.1 30.4 38.9 35.3 30.8

SSEM (%)

Major elements

Pb

NH3eN

Zn     

3.7 6.3 6.8 3.9 4.1

47.1 145.4 162.1 134.2 108.4

    

3.8 36.8 23.5 23.6 15.7

1.7 1.8 1.6 3.0 0.3

    

0.5 0.2 0.3 1.1 0.1

Available phosphorus 39.2 70.3 78.5 99.9 72.6

    

8.2 9.8 6.9 21.5 12.4

AVS (%)

Total nitrogen 1.9 2.2 2.8 3.1 2.1

    

0.5 0.4 1.3 0.5 1.1

0.4 1.1 1.1 1.3 1.3

    

0.4 0.2 0.3 0.4 0.1

13.1 40.4 49.1 38.5 22.0

    

5.6 9.2 8.9 10.3 6.2

V. Thiyagarajan et al. / Marine Environmental Research 70 (2010) 56e64

VHE

VH

VHW

0.6

1.5

PC

B

0.5

2 TN (%)

TOC (%)

TLC

A

2.5

59

0.4 0.3

1 0.2 0.5

0.1

0

0

C/S Ratio

6 5 4 3 2 1 0

D

0.12 Chl a (mg/g dry weight)

C

7

0.1 0.08 0.06 0.04 0.02 0

Summer Winter Summer Winter Summer Winter

Summer Winter Summer Winter Summer Winter

2004

2004

2004

2005

2005

2006

2006

2004

2005

2005

2006

2006

Fig. 2. Mean (n ¼ 6) sediment TOC (A), TN (B), C/S ratio (C) and Chlorophyll a (D) content for all five sites over the sampling dates. See Fig. 1 for site abbreviations.

or six sampling dates within each site (Thiyagarajan et al., 2006). However, all the four measured biochemical sediment parameters, total organic carbon (TOC), total nitrogen (TN), carbon/sulfur (C/S) ratio, and Chl a contents were significantly different among sites and sampling dates and there was a significant interaction between these two factors, sampling site and date (Fig. 2; twoway ANOVA; degree of freedom ¼ 12, 80; p < 0.05). The minimum TOC content was recorded at TLC (w0.3%) in winter 2005 and the maximum was recorded at VHE and VH (w2%) in summer 2005 (Fig. 2A). In most of the sampling dates, TOC at PC was significantly lower than that at VH. The TN content varied from w0.07% to 0.55% and showed seasonality that was characterized by slightly higher values in winter 2005 than other sampling dates at each site (Fig. 2B). The C/S ratio remained higher at TLC in five of the six sampling dates compared to other sites (Fig. 2C). Strikingly, C/S ratio in TLC varied dramatically between seasons and sampling years, i.e. it was w3 in 2004 winter but decreased to w1 in 2005 winter. The Chl a values also exhibited clear seasonality, characterized by the highest values in summer 2005 and the lowest values in winter 2004 (Fig. 2D). Interestingly, in winter 2005, the TOC content sharply declined at all sites except PC; the TN content reached the peak value at all sites; and the Chl a values declined dramatically from the previous sampling date again at all sites. The higher level of NH3, AVS and a negative value of redox potential in VH and VHW compared to TLC and PC indicated that the sedimentary environments in VH and VHW are highly anoxic (Table 1).

Changes in the sediment bacterial community composition at the five sites showed different temporal patterns (Fig. 4). However, there was no evidence for inter-annual changes of bacterial community in any particular site. For example, Fig. 4A, DeE showed a considerable overlap between winter and summer samples collected from TLC, VHW, and PC. In contrast, the bacterial communities in winter samples were distinctly separated from those in summer at VHE and VH (Fig. 4B and C).

3.2. Bacterial community analysis

3.5. Response of bacterial community to sewage discharge

All 120 samples were processed and analyzed at the same time (August 2006) to ensure consistency. Analysis of replicate PCR reactions from the same sediment sample produced highly reproducible profiles. The maximum number of T-RFs derived from samples was 110 (VHW in winter 2004), and the minimum was 50 (TLC in summer 2005). Visual observation of T-RFLP patterns and two-way crossed ANOSIM analysis among samples from the six sampling dates and the five sampling sites showed significant differences in the bacterial community composition for these two factors, sampling sites and date. Therefore, response of bacterial community to each factor by fixing the others was tested using MDS ordination.

VHW vs. PC. VHW represented a sewage discharge site and PC served as a control site. In order to verify if there was been an impact of sewage discharge on bacterial community in surface sediments, we used T-RFLP analysis and a pairwise comparison test (ANOSIM) to compare the bacterial communities at the two sites. Except in summer 2004 and winter 2005, the pairwise comparisons of the bacterial communities from VHW and PC showed low R values, indicating a high community resemblance (ANOSIM Global R values were <0.5, P > 0.05). The same trend was observed in the MDS ordination (Fig. 5A). Thus, there was no sign of adverse changes in the surface sediment bacterial community as a result of treated sewage discharge at VHW.

3.3. Spatial variations in bacterial community composition The MDS ordinations for each sampling date revealed a sitespecific pattern in the sediment bacterial community composition. In most of the sampling dates (except in winter 2005), the bacterial community at the VH formed a separate group on the lower right side of the MDS ordinations (Fig. 3AeC, EeF). The bacterial communities of the remaining four sites were loosely grouped. During winter 2005, the bacterial communities from three contrasting environments, TLC (oceanic side), VH (mid-harbour area) and PC (Pearl River side), formed a single group on left side of the ordination (Fig. 3D), whereas VHE and VHW had the least similarity. These results were supported by the ANOSIM results. Thus, the multivariate analysis indicated that the bacterial community composition changed in response to environmental pollution gradients. 3.4. Temporal variations in bacterial community composition

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A

Summer 2004

Stress :0.13

B Winter 2004

Stress :0.15

C

Summer 2005

Stress :0.18

D Winter 2005

Stress :0.1

E

Summer 2006

Stress :0.16

F Winter 2006

Stress :0.18

TLC

VHE

VH

VHW

PC

Fig. 3. MDS ordinations showing the spatial variations of the bacterial community compositions in sediments collected from six sampling dates (AeF). See Fig. 1 for site abbreviations.

3.6. Response of bacterial community to abatement of sewage discharge VHE vs. TLC. VHE represented a sewage abatement site and TLC served as the control site. These two sites had more or less the same water depth, salinity, temperature, and surface sediment composition during the sampling period. In all the six sampling dates, the pairwise comparisons of the sediment bacterial communities from TLC and VHE showed high R values, indicating low community resemblance (ANOSIM Global R values were >0.7, P < 0.01). The same trend was observed in the MDS ordination, in which TLC samples grouped at the upper left side, irrespective of sampling date, and the VHE samples at the lower right side (Fig. 5B). Thus, there was no sign of bacterial community recovery to pollution abatement at VHE. 3.7. Relationship between sediment characteristics and bacterial communities Table 2 shows the results of the regression multivariate analysis performed by the DISTLM forward test. Both the marginal and

conditional tests suggested a significant relationship between most of the observed sediment characteristics and bacterial community composition. Consequently, the constrained redundancy (RDA) ordination was obtained using DISTLM data. There was no clear trend or relationship between bacterial community and sediment characteristics when analyzed through season or sampling time (Fig. 6A). However, there were at least two trends (forming two bacterial community groups) in T-RFLP data that can be explained by the observed sediment characteristics. For instance, bacterial community composition between TLC and PC (Fig. 6B), between TLC and VHE (Fig. 6C) and between VHW and PC (Fig. 6D) are driven largely by the C/S ratio. 4. Discussion The overall goal of this study was to examine the spatio-temporal changes in the surface sediment bacterial community in response to environmental pollution gradients in a sub-tropical climate. The environmental pollution gradient in Victoria Harbour area led to marked shifts in the composition of bacterial communities as attested by the multivariate analysis of T-RFLP profiles (see Figs. 3e6).

V. Thiyagarajan et al. / Marine Environmental Research 70 (2010) 56e64

A

C

E

TLC

Stress : 0.19

B VHE

VH

Stress : 0.21

D

VHW

61

Stress : 0.16

Stress : 0.15

PC Stress : 0.14 Winter 2004

Summer 2004

Winter 2005

Summer 2005

Winter 2006

Summer 2006

Fig. 4. MDS ordinations showing the temporal variations of the bacterial community compositions in sediments collected from five sites (AeE). See Fig. 1 for site abbreviations.

The mid-harbour area (VH) showed the largest temporal changes in bacterial community composition when compared to the adjacent four sites. Surprisingly, bacterial communities at two contrasting environments (TLC vs PC) shared high similarity throughout the study period. Most importantly, there was no sign of adverse changes in bacterial community composition as a result of treated sewage discharge at VHW during the study period. One interesting feature highlighted in the present study was the fact that the sediment bacterial communities at the eastern site (TLC), nearest the Pearl River, shared high similarity with those at the western, oceanic site (PC) in all our sampling dates except in winter 2005. Because different pelagic-benthic environmental conditions (salinity and nutrient levels) presumably affect bacterial composition, we expected a seasonal shift in bacterial community composition at all sites and between sites along the environmental gradient. However, the two control sites, TLC and PC, were more heavily affected by seasonal changes in water quality (data not presented in this study but see Ho et al., 2008) and some sediment

conditions than were sites located within Victoria Harbour (such as VHE and VH). The lack of widespread seasonal shifts in the bacterial communities and the similarity between TLC and PC thus, raise the question of whether strong variations in environmental conditions in Hong Kong’s coastal area influence bacterial population in a measurable way. However, there was a significant relationship between sediment C/S ratio and bacterial communities in TLC and PC (Fig. 6B), even though there was a lack of temporal patterns (Fig. 4A and E). However, as expected, a striking seasonal trend was observed at the two central harbour sites, VH and VHE, i.e. summer samples formed a separate group at lower side in the MDS ordination (see Fig. 4B and C). Surprisingly, we did not observe such a clear seasonal trend at PC (site located close to Pearl River discharge area), where there was a strong seasonal gradient in salinity and water quality. At PC, it appeared that the seasonal variations in biochemical and water quality characteristics were too weak to create a significant seasonal shift in bacterial community pattern. On the other hand,

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V. Thiyagarajan et al. / Marine Environmental Research 70 (2010) 56e64

A

B

Stress : 0.17

VHW vs PC

Stress : 0.23

TLC vs VHE

Table 2 Tests of relationships between bacterial community composition at different sampling points (sites and times) and sediment characteristics (environmental conditions), using the non-parametric multivariate multiple regression analysis (DISTLM). P-values < 0.01 are in bold. TOM: Sediment Total organic matter; C/N ratio: carbon/nitrogen ratio; C/S: carbon/sulfate ratio; SEM: simultaneously extracted metals; AVS: Acid volatile sulphides. Marginal tests variables

Pseudo-F

P

Proportion of varience

Chl a Water content TOM C/N C/S Cu Pb Zn SSEM AVS Ammonium-nitrogen Total nitrogen

3.803 5.159 5.516 1.861 6.654 13.226 6.671 8.157 3.829 8.817 6.940 9.395

0.001 0.001 0.001 0.041 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001

0.031 0.042 0.045 0.016 0.053 0.101 0.054 0.065 0.031 0.070 0.056 0.074

Sequential tests

Pseudo-F

P

Proportion of varience

Cum. varience

Cu Ammonium-nitrogen C/S Total nitrogen AVS Zn Chl a Water content TOM C/N Pb SSEM

13.226 5.044 4.845 4.757 3.153 3.335 2.568 1.447 2.134 1.110 1.041 0.279

0.001 0.001 0.001 0.001 0.004 0.008 0.042 0.133 0.013 0.345 0.415 0.988

0.179 0.037 0.035 0.033 0.021 0.022 0.017 0.009 0.014 0.007 0.007 0.002

0.109 0.138 0.173 0.206 0.227 0.248 0.266 0.276 0.291 0.296 0.303 0.305

Significant values (P < 0.05) are showed in bold values.

TLC

VHE

VHW

PC

Summer 2004 Winter 2004 Summer 2005 Winter 2005 Summer 2006 Winter 2006

Fig. 5. MDS ordinations comparing the sediment bacterial communities between (A) sewage discharge site (VHW) and control site (PC) over six sampling dates, and (B) sewage abatement site (VHE) and control site (TLC) over six sampling dates. See Fig. 1 for site abbreviations.

the nutrient concentrations (e.g. nitrate, ammonium, and phosphate) of water column in Victoria Harbour (especially at VH and VHE) were much higher than either side of the harbour, especially in summer, indicating the influence of sewage effluent (Yin and Harrison, 2007). Such specific seasonal gradient in water quality characteristics might have formed a clear seasonal shift in bacterial community on surface sediment in these two sites. The bacterial community at the mid-harbour site, VH, was distinct from the adjacent four sites in all sampling dates except in winter 2005. This observation was not surprising since previous studies have reported substantial shifts in the bacterial community composition in response to hydrocarbons (Cuny et al., 2007), xenobiotic (Hogan and Ward, 1998), and heavy metal (Ford et al.,

2005; Gillan et al., 2005; Ogilvie and Grant, 2008) contamination. Although direct discharge of pollutants and untreated sewage into VH has been dramatically reduced since December 2001, this site still has elevated organic content, heavy metals, and organic pollutants (see Table 1) and is highly anoxic with low electrochemical potential. Alternatively, site-specific variation in macrobenthic communities may also explain patterns of bacterial community composition. For instance, deposit-feeding opportunistic polychaete species such as Capitella spp. were numerically dominant at VH when compared to the adjacent four sites (Thiyagarajan et al., 2006). Deposit-feeding activities substantially affect spatial heterogeneity in bacterial community composition in temperate coastal sediments (Plante and Wilde, 2004). Unfortunately, no information is currently available on the specific effect of bioturbation by deposit-feeding opportunistic polychaete worms on the structure of bacterial communities in organically enriched sediments, especially in sub-tropical regions. Nevertheless, the effect of organic enrichment on bacterial communities appears to be site-specific. In contrast, a recent field survey near an open ocean fish farm on the southeast coast of Oahu, Hawaii showed that organic enrichment did not significantly affect the bacterial community composition there (Yoza et al., 2007). The lack of similarity in the bacterial community compositions between TLC and VHE was unexpected as the discharge of a large amount of untreated sewage at VHE has been dramatically reduced since 2001. Noticeably, water quality (i.e. low number of Escherichia coli, nutrient content and Chl a levels) at VHE has improved significantly after the implementation of the Harbour Area Treatment Scheme (HATS) in 2001 and the toxic metal discharges into the VHE have also been reduced (HKEPD, 2006). As a result, several sedimentary biochemical parameters and pollution levels have shown a decreasing trend (Li XD, personal communication).

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Fig. 6. Redundancy analysis (RDA) ordination of the T-RFLP data from R2 model with sediment (environmental) variables. Percentage of variation explained by individual axes relate to the percentage explained out of the fitted model are reported. See Fig. 1 for site abbreviations. S: summer; W: winter; numbers next to each site refer to sampling time.

However, long-term simultaneous monitoring on sediment bacterial community, biochemical characteristics and pollution loading is required to address the bacterial community recovery at VHE. In the vicinity of sewage outfalls, VHW, sediment organic carbon was expected to be increasing. Although TOC content at this site was slightly higher than that at the control sites, PC and TLC, it was lower than that at central harbour sites, VH and VHE. The observed slight increase in TOC content was therefore not expected to lead to the changes through time in the bacterial community composition at VHW. This was the case in this study. The unexpected sudden changes in sedimentary biochemical characteristics, especially TOC, TN, and Chl a contents (see Fig. 2), in one of the sampling dates (winter 2005) have dramatically altered the spatial pattern of bacterial community structure; VH, TLC, and PC were more similar, while distinctive communities were observed at both VHW and VHE. Such dramatic spatial shift in bacterial community composition could be attributed to the observed seasonal variation in sedimentary characteristics and to their

interactive effects on specific bacterial groups. These results suggest that the bacterial communities were spatially structured in the study area by sedimentary variables directly linked to nitrogen, carbon and phytoplankton content along the pollution gradient. Therefore, seasonal shift in water quality play a role in the structure of the resulting bacterial communities in sediments and presumably have impacts on microbial function. Although we did not study bacterial community gene and protein expression, it would be interesting to see how their levels are correlated to the sedimentary factors and to the identities of the dominant bacterial populations identified in the sediments. In this study, T-RFLP technique has been shown to be sensitive and effective for detecting changes in bacterial community composition in response to different kinds of environmental and anthropogenic disturbances. Coupling molecular tools with traditional measurements of physico-chemical parameters thus, allows us to evaluate the response of micro-benthos to anthropogenic disturbances. Among a variety of molecular tools, we started with T-RFLP due to its simple operation with an automatic genetic

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analyzer. Nevertheless, T-RFLP analysis does not allow the identity of bacteria in the samples. Investigators who are interested in the identification of bacteria in samples need to choose other techniques, such as denaturing gradient gel electrophoresis (DGGE) (Muyzer et al., 1993), which allows the extraction and sequence of specific bands resolved on the gel. It shall be noted that fingerprinting techniques such as T-RFLP and DGGE may not detect minority bacterial populations since template DNAs from these populations represent a small fraction of the total extracted DNA and may not be amplified by PCR due to kinetic bias (Liu et al., 1997, 2008). In conclusion, this study demonstrated that surface sediment contaminants together with sediment biochemical characteristics and overlying water quality conditions can be quite important in structuring bacterial communities in soft-bottom subtropical coastal habitats. The T-RFLP analysis appeared to be an effective tool for assessing benthic bacterial community responses to coastal pollution and environmental changes. Our data provide a link between bacterial community information and the effects of environmental pollution gradient on the structural qualities of benthic bacterial commentaries in coastal ecosystems. Acknowledgements The authors thanks Dr. Xiang Dong Li at The Hong Kong Polytechnic University for the sharing of the biochemical characteristics and pollution levels data. We wish to thank the two anonymous reviewers, whose critical and constructive comments strengthened this paper. We also wish to thank Dr. Shawn M. Arellano for her valuable comments and for critically reading the manuscript. The work described in this paper was funded by the Area of Excellence Scheme under the UGC of the Hong Kong SAR (Project no. AoE/ P-04/04-2-II). References Anderson, M.J., 2003. DISTLM Forward: A FORTRAN Computer Program to Calculate a Distance-Based Multivariate Analysis for a Linear Model using Forward Selection. Department of Statistics, University of Auckland, New Zealand. Bissett, A., Bowman, J.P., Burke, C., 2006. Bacterial diversity in organically-enriched fish farm sediments. FEMS Microbiology Ecology 55, 48e56. Clark, K.R., Gorley, R.N., 2001. PRIMER V5: User Manual/Tutorial. PRIMER-E Ltd, Plymouth, United Kingdom. Clarke, K.R., Warwick, R.M., 1994. Change in Marine Communities: An Approach to Statistical Analysis and Interpretation. Natural Environment Research Council, Plymouth, UK, pp. 144. Cuny, P., Miralles, G., Cornet-Barthaux, V., Acquaviva, M., Stora, G., Grossi, V., Gilbert, F., 2007. Influence of bioturbation by the polychaete Nereis diversicolor on the structure of bacterial communities in oil contaminated coastal sediments. Marine Pollution Bulletin 54, 452e459. Duran, R., Ranchou-Peyruse, M., Menuet, V., Monperrus, M., Bareille, G., Gon, M.S., Salvado, J.C., Amouroux, D., Guyoneaud, R., Donard, O.F.X., Caumette, P., 2008. Mercury methylation by a microbial community from sediments of the Adour Estuary (Bay of Biscay, France). Environmental Pollution 156, 951e958. Edwards, U., Rogall, T., Helmut, B., Emde, M., Böttger, E., 1989. Isolation and direct complete nucleotide determination of entire genes. Charcterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Research 17, 7843e7853. Ford, T.E., Jay, J., Patel, A., Kile, M., Prommasith, P., Galloway, T., Sanger, R., Smith, K., Depledge, M., 2005. Use of ecotoxicological tools to evaluate the health of New Bedford Harbor sediments: a microbial biomarker approach. Environmental Health Perspectives 113, 186e191. Forthin, N., Beumier, D., Lee, K., Greer, C.W., 2004. Soil washing improves the recovery of total community DNA from polluted and high organic content sediment. Journal of Microbiology Methods 56, 181e191. Gillan, D., Danis, B., Pernet, P., Joly, G., Dubois, P., 2005. Structure of sedimentassociated microbial communities along a heavy-metal contamination gradient in the marine environment. Applied Environmental Microbiology 71, 679e690. Han, J.B., Mu, Y.L., Wang, L.M., 1999. Advances in research of marine aquaculture and coastal water pollution. Fish Science 18, 40e43.

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