Spatial–temporal variations and diversity of the bacterioplankton communities in the coastal waters of Kuwait

MPB-07173; No of Pages 11 Marine Pollution Bulletin xxx (2015) xxx–xxx

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Spatial–temporal variations and diversity of the bacterioplankton communities in the coastal waters of Kuwait Awatef Almutairi Kuwait University, Faculty of Science, Department of Biological Sciences, P.O. Box 5969, Safat 13060, Kuwait

a r t i c l e

i n f o

Article history: Received 14 January 2015 Received in revised form 23 August 2015 Accepted 15 September 2015 Available online xxxx Keywords: Bacterioplankton DGGE Kuwait Proteobacteria Rhodobacteraceae Alteromonadaceae

a b s t r a c t The dynamics and composition of the bacterial community in the coastal waters of Kuwait are poorly understood. In this study, the spatial–temporal variations in the bacterial composition in the surface water along the Kuwaiti coast was examined by 16S rRNA denaturing gradient gel electrophoresis (DGGE) fingerprinting and phylogeny analyses. The sampling sites were Kuwait Bay, Al-Sabbiya (north of the bay) and Al-Khairan (to the south). The bacterial composition was more variable in the summer for all sites. A cluster analysis of the DGGE fingerprint revealed two main clusters, indicating a temporal similarity between sites. Kuwait Bay and Al-Khairan were more similar to each other than to Al-Sabbiya. The bacterial community composition exhibited distinctive spatial variations, with more diversity at Al-Khairan and less diversity at Al-Sabbiya. At all sites, the dominant bacteria were Alphaproteobacteria, in particular Rhodobacteraceae, followed by Alteromonadaceae (Gammaproteobacteria) and Bacteroidetes. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction The marine environment of Kuwait is situated at the western edge of the northern part of the Arabian Gulf. Kuwaiti waters are shallow, with a maximum depth of approximately 30 m (Khalaf et al., 1982). The coastal habitats of Kuwait range from exposed beaches to rocky highland, and sand silt and clay exist in both the intertidal and subtidal littoral zones (Al-Nafisi et al., 2009). Kuwaiti waters are characterized by a salinity gradient from north to south due to the diluting influence of the fresh water inflow from Shatt Al-Arab River, north of the Arabian Gulf. The water column is generally well mixed year-round, and the waters are generally oxygen-rich (Al-Yamani et al., 2004). The water temperature reaches its highest value during summer; however, the mean annual temperature of water is 23.8 °C (Dames and Moore, 1983). The mean pH is 8.2, with no significant seasonal variation. The settling of suspended matter from river discharge and dust storms dramatically affects the turbidity levels of the water column. The turbidity levels are extremely high at the north side, but decline dramatically toward the south (Al-Ghadban and El-Sammak, 2005; Al-Enezi et al., 2014). Kuwait waters are generally nutrient-rich, with high biological productivity (Al-Yamani, 2001). The inflow from Shatt Al-Arab, sewage discharge, and land runoff have a major influence on the hydrodynamics, water quality, and, most importantly, the biological productivity of the northern part of the Arabian Gulf (Al-Yamani et al., 2004).

E-mail address: [email protected].

The physicochemical characteristics, circulation, and geomorphology of Kuwaiti waters have been thoroughly examined; nevertheless, little is known about the bacterioplankton communities in these waters. Very few studies have focused on the abundance of bacterioplankton in the Kuwait marine environment. The bacterioplankton abundance was high throughout Kuwaiti waters, with an average of 3.18 × 106 cells/ml (Al-Yamani et al., 1997). This reflects the wellmixed nature of Kuwaiti waters and the northwestern Arabian Gulf, which was also found to have an average bacterioplankton abundance of 1.73 × 106 cells/ml (Al-Rifaie et al., 2008). Marine bacterioplankton are the major drivers in the biogeochemical processes in aquatic ecosystems (Kent et al., 2007; Pomeroy et al., 2007). Bacterioplankton facilitate the cycling of organic matter and nutrients by maintaining the marine ecosystem's health, balance, and ability to recover from damage. Marine bacterial populations are usually complex and often contain unidentified or uncultivated members (Pace, 1997). In addition, marine bacteria exhibit a seasonal diversity pattern in pelagic ecosystems, showing a higher diversity during winter than summer (Fuhrman et al., 2006). The bacterioplankton abundance and diversity in the marine environment is regulated by numerous environmental factors, such as temperature, salinity, dissolved oxygen levels and nutrient availability (Fuhrman et al., 2006; Gilbert et al., 2009). The coastal waters of Kuwait are severely stressed by marine pollution, the main sources of which are petroleum-related industries, urbanization, industrial expansion, municipal wastewater, effluent from desalination and power plants, urban runoff, and the increase in marine-based recreation (Al-Muzaini, 2002; Al-Yamani et al., 2006).

http://dx.doi.org/10.1016/j.marpolbul.2015.09.016 0025-326X/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article as: Almutairi, A., Spatial–temporal variations and diversity of the bacterioplankton communities in the coastal waters of Kuwait, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.09.016

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The major pumping failure at the Mishref sewage station in 2009 released approximately 180,000–200,000 m3/day of untreated sewage into the coastal environment for several months, and the sewage discharged into the Kuwaiti coastal waters was usually high in organic content (Ghannoum et al., 1991; Al-Ghadban et al., 2002). Sewage discharge may cause coastal eutrophication, the accumulation of toxic substances in coastal sediments and support the growth of hazardous algal blooms. This study aims to examine the seasonal spatiotemporal patterns of bacterioplankton dynamics in the surface water at three areas along the coast of Kuwait to gain a deeper insight into the microbial ecology of this more impacted marine environment. The diversity of the bacterial community will also be explored to gain an overall understanding of the structure of the bacterioplankton in these waters and the effect of various environmental factors on modulating the bacterial community structure in Kuwaiti waters.

Sampling was conducted every 2 months from April 2010 until February 2011. A 1-L sample of surface seawater was collected in a clean container at each site. The samples were placed in a cooler to maintain their low temperature and were immediately transported to the laboratory. At the same time that the samples were collected, the ambient water temperature, salinity, pH, turbidity, and dissolved oxygen content of the surface water were measured directly at the site using a Water Quality Checker U-10 (HORIBA). In the lab, the samples were prefiltered through a 47-mm-diameter polycarbonate filter (nominal pore size 3 μm) to remove the large particles and then filtered through a 0.22-μm PES filter (Nalgene). The filters were then immediately placed in sterile containers and stored at −20 °C before being processed. The chlorophyll −a, total organic carbon, petroleum hydrocarbons and nutrient measurements were obtained from the annual statistical bulletin of the environment published by the Kuwait Environmental Public Authority (KEPA).

2. Materials and methods

2.2. DNA extraction

2.1. Study site and sampling

Under sterile conditions, the total genomic DNA was extracted from the filters. Each sample manipulation was performed separately to avoid cross-contamination. The DNA was extracted from frozen filters using a FastDNA spin kit for soil (MP Biomedical) according to the manufacturer's instructions. The integrity of the extracted DNA was verified by electrophoresis on a 0.8% agarose gel in 1× TAE buffer.

The water samples were collected from three sites (Fig. 1). The northernmost site was Khor Al-Sabbiya (N29.37.133, E48.09.099), a long submerged estuarine channel located on the northern coast of Kuwait. Al-Sabbiya is a muddy intertidal flat that is characterized by a plume of suspended sediments. The second site was Kuwait Bay, an elliptical embayment at the northwest corner of the Arabian Gulf and north of Kuwait city (N29.19.429, E47.52.774). Kuwait Bay is a semienclosed, nonestuarine environment with an area of approximately 750 km2 (Al-Ghadban and El-Sammak, 2005). The water is shallow throughout, with an average depth of approximately 8 m and a maximum depth of less than 15 m (Al-Yamani et al., 2004). Kuwait Bay has soft sediments, slow tidal currents, high turbidity, little sediment transport, and the bay has undergone considerable development and is almost completely urbanized. The third site is Al-Khairan, on the southern coast of Kuwait (N28.32.820, E48.28.144). South of Kuwait Bay, the coast is dominated by sand beaches that are relatively unprotected from the open Gulf waves, and almost the entire south coast has been anthropogenically modified.

2.3. Polymerase chain reaction (PCR) amplification The 16S rRNA genes were amplified from the DNA templates by the Ready-to-Go PCR beads (GE Healthcare, UK) using the universal bacterial primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) (Lane, 1991). PCR was performed in 25-μL reaction mixtures (0.5 μM of each primer and 1 μL of the DNA template). The cycling conditions were 94 °C for 5 min, followed by 30 cycles of 94 °C for 1 min and 55 °C for 1 min, with a final extension at 72 °C for 10 min. The amplified DNA was diluted by a factor of 100 and used as a template for a second PCR amplification to amplify the 590-bp DNA fragments of the V3 region of the 16S rRNA using the primers GM5F (5′-CCTACGGGAGGCAGCAG-3′) and 907R (5′-CCGTCA

Fig. 1. Map of Kuwait showing the three sampling sites.

Please cite this article as: Almutairi, A., Spatial–temporal variations and diversity of the bacterioplankton communities in the coastal waters of Kuwait, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.09.016

A. Almutairi / Marine Pollution Bulletin xxx (2015) xxx–xxx

ATTCMTTTGAGTTT-3′) (Schafer and Muyzer, 2001). A triplicate series of PCR was performed for each sample. The amplification was performed at 94 °C for 5 min, followed by 25 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, and a final extension at 72 °C for 30 s. The PCR products were pooled and purified using a PCR purification kit (Qiagen). 2.4. PCR-denaturing gradient gel electrophoresis (DGGE) analysis PCR-DGGE analysis of the diluted amplified 16S rRNA genes was performed under the same conditions as described earlier using the GM5FGC clamped forward primer and the 907R reverse primer (Schafer and Muyzer, 2001), and the PCR products were purified using a PCR purification kit (Qiagen). DGGE analysis was performed using the Dcode Universal Mutation Detection system (Bio-Rad). The PCR products (350 ng) from each sample were run on a 6% polyacrylamide gel with a denaturant gradient of 35–55% (100% is defined as 7 M urea plus 40% deionized formamide). Electrophoresis was performed in 1× TAE buffer (2 M Tris base, 1 M acetic acid, and 50 mM EDTA, for 50× stock solution) at a constant voltage of 50 V and a temperature of 60 °C for the optimal duration of 16 h. The separated PCR products were stained for 30 min in the dark with SYBR Green I (Molecular Probe), visualized, and photographed. The most prominent bands (bands with high intensity) were excised directly from the gel and eluted in 100 μL of ultrapure Milli-Q water overnight at 4 °C. The eluted DNA was re-amplified using the universal primers GM5F-GC and 907R and inspected by a second DGGE analysis. The excised and purified individual bands from the second DGGE were re-amplified using the primers GM5F and 907R, without the GCclamp, for sequencing. The Binary Matrices resulting from the DGGE banding pattern were analyzed using cluster analysis (SPSS version 18).

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Sequencing Kit (Applied Biosystems, USA). The sequencing reactions were analyzed with a 3130 ×L Genetic Analyzer (Applied Biosystems, USA). All sequences from the clone libraries were screened for vector contamination using the VecScreen tool available at the NCBI site (http://www.ncbi.nlm.nih.gov/tools/vecscreen). The correct sequences were then checked for chimeric structures using the DECIPHER program (Wright et al., 2012) (http://decipher.cee.wisc.edu/index.html). The taxonomic identities of the checked sequences were assessed using the Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm. nih.gov/BLAST) against the nucleotide database available from GenBank and verified using the ribosomal Database Project classifier (http://rdp. cme.msu.edu). 2.8. Phylogenetic analysis The sequences of randomly selected representative clones and their closest neighbors from GenBank were aligned and manually edited using the multiple-alignment program ClustalX2. A phylogenetic tree was constructed by the neighbor-joining method, based on the Maximum Composite Likelihood option in MEGA 6.0 (Tamura et al., 2013). The branches of the resulting tree were evaluated by a bootstrap analysis based on 2000 replicates. 2.9. Nucleotide sequence accession number The sequences have been deposited in GenBank under the accession numbers KP262538 to KP262637 for the DGGE bands and KP262638 to KP262868 for the clone libraries. 3. Results

2.5. Clone library preparation

3.1. Environmental parameters of the study sites

Clone libraries were constructed from the August 2010 samples to expand the results from the DGGE analysis. The purified PCR products from each site were cloned into the pGEM-T cloning vector (Promega). The ligation products were used to transform chemically competent DH5-α Escherichia coli cells. Approximately 100 positive clones were randomly selected from each library and the plasmids were isolated using the Qiaprep Spin Miniprep kit (Qiagen).

During the study period, the recorded temperatures at all sites exhibited the expected summer–winter differences, with the minimum temperatures during the winter. The surface water temperature was 18–31.2 °C in Al-Sabbiya (Table 1), 11.2–31.4 °C in Kuwait Bay, and 16–32.4 °C in Al-Khairan (Table 1). The results show slight spatial variations in temperature among the three sites; the temperature tended to increase from north to south, with the highest annual average surface water temperature at Al-Khairan (25.5 °C) and the lowest at AlSabbiya (24.9 °C). The surface water pH also showed slight spatial variations, with the lowest seasonal pH values at Al-Sabbiya (pH = 8.18) and increasing pH further south at Al-Khairan (pH = 8.29). However, the pH was higher at all sites during the summer. The salinity of the

2.6. Statistical analysis The coverage of the constructed 16S rRNA libraries was calculated according to the formula C = [1 − (n/N)] (Good, 1953), where n is the number of sequence types that occur only once in the library and N is the total number of clones examined. The Shannon diversity index of the constructed clone libraries was calculated according to the Shannon Index (Shannon and Weaver, 1949) using the formula H = (−∑Pi* ln Pi), where H is the Shannon diversity index, Pi is the proportion (n/N) of individuals of one particular species (n) divided by the total number of individuals (N), and ∑ is the sum of all species identified. To measure the evenness of a community, the Shannon Evenness Index (SEI) was calculated using the formula SEI = H / lnS, where H is the Shannon diversity index and S is the number of species encountered in a sample. The Chao-1 nonparametric species richness estimator of the constructed clone libraries was calculated based on the formula Sest = Sobs + (a2 / 2b) described by Colwell and Coddington (1994), where Sest is the estimated richness, Sobs is the observed number of species, a is the number of sequences observed only once (i.e., singletons) and b is the number of sequences observed only twice (i.e., doubletons). 2.7. Sequence analysis The DGGE-purified PCR amplicons and positive clones from each library were sequenced using the Big Dye Terminator V3.1 Cycle

Table 1 Environmental parameters of the sampling sites. Site

Month

Temperature (°C)

pH

Salinity (%)

Dissolved oxygen (mg/L)

Al-Sabbiya

April 2010 June 2010 August 2010 October 2010 December 2010 February 2011 April 2010 June 2010 August 2010 October 2010 December 2010 February 2011 April 2010 June 2010 August 2010 October 2010 December 2010 February 2011

27.5 31.0 31.2 30.0 12.0 18.0 27.5 31.0 31.4 29.4 20.1 11.2 27.5 30.0 32.4 28.1 19.0 16.0

8.21 8.12 8.28 8.13 8.12 8.22 8.36 8.36 8.32 8.18 8.22 8.22 8.39 8.34 8.33 8.36 8.12 8.22

3.59 3.61 3.48 3.46 3.47 3.67 3.89 3.89 3.88 3.82 3.89 3.82 3.89 3.99 3.89 3.92 3.98 3.87

6.01 5.98 5.01 5.46 7.9 7.43 5.99 5.66 5.09 5.78 6.06 7.07 6.01 5.99 4.98 5.63 6.68 7.32

Kuwait Bay

Al-Khairan

Please cite this article as: Almutairi, A., Spatial–temporal variations and diversity of the bacterioplankton communities in the coastal waters of Kuwait, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.09.016

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water column was lowest at Al-Sabbiya (3.46–3.67%), followed by Kuwait Bay (3.82–3.89%) and Al-Khairan (3.87–3.99%). The dissolved oxygen concentration at all sites was lower in the summer and varied between 5.01 and 7.93 mg/L at Al-Sabbiya, 5.66 and 7.07 mg/L at Kuwait Bay, and 4.98 and 7.32 mg/L at Al-Khairan. The chlorophyll-a levels (Table 2) exhibited a slight variation throughout the study period at Ras Ajouzah (Kuwait Bay area) and increased in the summer at Ras Al-Zour (located at a close vicinity to the Al-Khairan area). The total organic carbon content at the abovementioned sites was higher during spring and summer, with a noticeable reduction in early summer (June). The average total organic carbon concentration during the study period was higher around the Al-Khairan area. The two sites exhibited slight seasonal differences in nutrient concentrations. The dissolved ammonium levels (NH+ 4 -N) in water were high during summer, and the levels of nitrate (NO3-N) and nitrite (NO2-N) also increased dur− − ing summer. The levels of NH+ 4 , NO3 and NO2 were higher at the Ras Al-Ajouzah (Kuwait Bay area) site compared to Ras Al-Zour. The total phosphorus (PO− 4 P) levels in the Kuwait Bay area increased during summer, whereas at the Al-Khairan area, an increase in the total phosphorus (PO− 4 P) levels can be seen during late summer to winter. However, the total phosphorus (PO4-P) concentration was higher around Al-Khairan area. The petroleum hydrocarbon levels increased at both sites during summer, and were, in most cases, higher around the Al-Khairan area compared to Kuwait Bay. 3.2. DNA fingerprint of the bacterial community dynamics The DGGE profiles retrieved from the study sites suggested that the bacterial community showed seasonal variations (Fig. 2). The band pattern was reproducible and some bands were common in almost all samples, while others were only present during certain months. The number of bands obtained was similar among samples (10 to 15) and was similar among study sites, indicating high bacterial richness at all sites. The changes in the banding patterns occurred from summer (June–August) to autumn (October) in all study sites, with the number of bands and their intensity clearly increased, which indicated the higher bacterial richness of the study sites at that time. To better visualize the relationships of the bacterial species within each site and between sites, a cluster analysis of the obtained DGGE fingerprints was constructed (Fig. 2). The cluster analysis between sites revealed pronounced temporal and spatial variations, appearing in two main clusters. The larger cluster showed a similarity in the banding pattern between the study sites during the summer with three sub-clusters evident from the analysis. The first sub-cluster showed a similarity in the banding pattern of Kuwait Bay samples collected from summer samples (June–August). The second sub-cluster showed a similarity in the banding pattern among Al-Khairan samples collected from summer (June–August) and Autumn (October–December). The other

sub-cluster showed a similarity in the banding patterns of the AlSabbiya samples collected from April to December (spring to autumn). Cluster II showed a similar banding pattern among the three sites in the colder season (December, February, and April), with two distinct sub-clusters. In this cluster, the Kuwait Bay samples from April and December were clustered separately from the other colder season samples of the other two sites. The results show a distinctive spatial pattern between sites in the warmer season, in which the three sites form three separate clusters. However, the Kuwait Bay and Al-Khairan samples were more similar to each other than to Al-Sabbiya, whereas during the colder season, all study sites showed more similarity to one another. After sequencing, a total of 100 sequences were obtained. The phylum composition of the sequence bands and their relative abundance are shown in Fig. 3. More than 60% of the sequences were Alphaproteobacteria that belonged to the Rhodobacteraceae family, and 8% of the sequences belonged to the SAR11 cluster. Interestingly, those sequences were detected in all samples analyzed from the three study sites. The second most represented phylogenetic group of sequences belonged to the Gammaproteobacteria group (26%). Of these, the majority belonged to the Aletromonadaceae family; however, some sequences belonged to the Pseudoalteromandaceae, Vibrionaceae, and Alcanivoraceae families. Six sequences belonging to the Bacteroidetes and Verrucomicrobia phyla were found at AlKhairan, and sequences belonging to the Actinobacteria phylum were found only at Al-Sabbiya. 3.3. Diversity and classification of the bacterial 16S rRNA gene clone library sequences To obtain a more detailed analysis of the bacterial community structure of the study sites, clone libraries were constructed from the August 2010 samples, which showed higher intensities on a DGGE gel. A total of 231 clones from the three sites were sequenced. The coverage of the constructed libraries was high, ranging from 93% to 97% (Table 3), which indicated that each clone library displayed the diversity of the sample site. The Shannon diversity index and Chao1 estimators of species diversity were calculated for each clone library (Table 3) and showed lower biodiversity indices at Al-Sabbiya than at Kuwait Bay or Al-Khairan. However, the evenness of species abundance at Al-Khairan was lower than at Kuwait Bay or Al-Sabbiya. The Chao1 estimator for species richness values were highest at Al-Khairan, followed by Kuwait Bay, suggesting that the bacterial community at Al-Sabbiya in the summer was composed of few phylotypes, whereas the communities at Al-Khairan and Kuwait Bay exhibited higher levels of species richness. The obtained clone sequences primarily belonged to the phyla Proteobacteria and Bacteroidetes. The phylum composition and relative

Table 2 Chlorophyll-a, nutrients and pollutant parameters obtained from the annual statistical bulletin of environment published by Kuwait Environmental Public Authority (KEPA) for Ras Ajouzah (Kuwait Bay area) and Ras Al-Zour. Site

Month

Chl-a (mg/L)

TOC (mg/L)

PHC'S (μg/L)

PO4-P (μg/L)

NH+ 4 -N (μg/L)

NO2-N (μg/L)

NO3-N (μg/L)

Ras Ajouzah (Kuwait Bay area)

April 2010 June 2010 August 2010 October 2010 December 2010 February 2011 April 2010 June 2010 August 2010 October 2010 December 2010 February 2011

1.000 1.000 1.000 1.000 1.400 0.870 0.970 0.940 1.300 0.900 1.000 1.000

2.26 1.20 2.33 2.309 2.341 2.105 4.178 1.270 2.300 1.675 – 1.879

1.610 0.594 1.484 0.551 0.850 0.271 2.750 0.622 1.028 1.429 – 0.547

26.4 9.540 37.1 14.5 26.00 13.320 28.170 7.310 14.60 157.90 – 72.030

260.00 70.00 690.00 148.00 3.00 326.00 110.00 349.00 140.00 275.00 – 246.00

7.750 0.00 2.60 3.90 7.60 0.750 0.600 1.620 3.500 1.800 – 0.480

0.130 1.240 12.800 2.100 7.400 15.820 0.410 0.00 3.40 0.00 – 13.320

Ras Al-zour

Chl-a: Cholorophyll-a, TOC: Total Organic Carbon, PHC's: Petroleum Hydrocarbons, PO4-P Phosphate, NH+ 4 -N: Ammonia and total nitrogen, NO2-N : Nitrite, NO3-N : Nitrate. –No measurement taken.

Please cite this article as: Almutairi, A., Spatial–temporal variations and diversity of the bacterioplankton communities in the coastal waters of Kuwait, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.09.016

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Fig. 2. Seasonal comparison of bacterioplankton communities at Al-Sabbiya, Kuwait Bay, and Al-Khairan, from April 2010 to February 2011. (A) Denaturing gradient gel electrophoresis (DGGE) band profiles of the 16S rRNA gene amplified using the total genomic DNA extracted from water samples collected from Kuwait Bay (Lanes 1–6), Al-Sabbiya (Lane 7–12), and Al-Khairan (Lane 13–18). (B) 16S rRNA-gene based cluster dendrogram representing the clustering profile.

abundances of the individual libraries are shown in Fig. 4. A total of 166 sequences were classified to the Proteobacteria phylum and five bacterial subphyla were identified: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, and Epsilonproteobacteria. The two most predominant subphyla were Alphaproteobacteria and Gammaproteobacteria, whereas the others were less dominant

and were not detected at all study sites. Phylogenetically, 83 clones (36.4%) belonged to the Alphaproteobacteria, 70 clones (30.7%) belonged to the Gammaproteobacteria, and 56 clones (24.56%) belonged to the Bacteroidetes phyla. The remaining 22 clones (9.5%) belonged to other bacterial groups (Actinobacteria, Verrucomicobia, Betaproteobacteria, Epsilonproteobacteria, and unclassified bacteria).

Fig. 3. Temporal and Spatial changes in the relative abundance of the bacterial community structure based on prominent DGGE bands sequencing from the study sites.

Please cite this article as: Almutairi, A., Spatial–temporal variations and diversity of the bacterioplankton communities in the coastal waters of Kuwait, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.09.016

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Table 3 Analysis of the bacterial clone libraries from the study sites. Sample site

No. of sequenced clones

Coverage (%)

Shannon diversity index (H)

Shannon equitability (EH)

Chao1

Kuwait Bay Al-Sabbiya Al-Khairan

79 77 75

93.67 94.8 97.33

2.82 2.64 2.88

0.941969 0.933929 0.886516

40.25 33.00 40.40

The Alphaproteobacteria were more abundant in Kuwait Bay (42%) and Al-Sabbiya (43%). Bacteroidetes were the major phylum found at Al-Khairan (43%) and least abundant in Kuwait Bay (9%). Gammaproteobacteria composed the major group in Kuwait Bay (38%) and their presence varied in Al-Sabbiya and Al-Khairan (25% and 28%, respectively). The Actinobacteria abundance was very low at Al-Sabbiya and Al-Khairan and was not detected in Kuwait Bay. The less predominant sequences were related to Betaproteobacteria, Deltaproteobacteria, and Epsilonproteobacteria. Verrucomicrobia and unclassified bacteria sequences were detected as minor groups in the clone libraries and were not found at all the sample sites. Within the predominant Alphaproteobacteria subphylum, 56% of the sequences were closely related to the bacteria genera that are frequently found in marine environments (e.g., Roseovarius, Thalassobius, Marivita, and Hyphomonas) and 6% of the sequences were closely related to Candidatus pelagibacter, the SAR11 cluster representative (Fig. 5). Furthermore, 37.5% of the sequences belonged to unclassified

Alphaproteobacteria sequences that are commonly found in freshwater and marine habitats. The sequences of the second major subphylum, Gammaproteobacteria, were closely related to the Alteromonas, Oceanospirillum, and Pseudoalteromonas genera. Representatives of the order Legionellales, such as Legionella and the obligate intracellular bacterial pathogen Coxiella, were found at Al-Khairan, along with a number of sequences belonging to unclassified Gammaproteobacteria. In addition, 50.9% of the Bacteroidetes phylum sequences were closely related to the unclassified Bacteroidetes found in marine environments. However, the classified sequences (Owenweeksia and Gilvibacter) were related to seawater isolates that belonged to the subphyla Flavobacteriia (Riedel et al., 2012; Yoshizawa et al., 2012). Nine of the classified Bacteroidetes sequences belonged to the subphylum Sphingobacteriia and were related to soil isolates of the genus Lewinella (Buerger et al., 2012). 3.4. Phylogenetic analysis of the bacterial 16S rRNA genes across all study sites A phylogenetic tree was constructed to show the relationships between the most dominant sequence clones from the study sites (32 sequences representing 231 sequences) and their closest neighbors (Fig. 6). The phylogenetic tree of the dominant sequences did not include any of the sequences that had very low abundance (b20%) or the unidentified bacterial sequences. The two phyla identified as having the majority of the sequences were classified as Proteobacteria and Bacteroidetes. Twenty-four out of the 33 analyzed clones fell into the two predominant

Fig. 4. Pie charts of the relative abundance of obtained bacterial 16S rRNA clones. A. All sequenced clones from all study sites; B. Al-Sabbiya bacterial 16S rRNA clones; C. Kuwait Bay bacterial 16S rRNA clones; D. Al-Khairan bacterial 16S rRNA clones.

Please cite this article as: Almutairi, A., Spatial–temporal variations and diversity of the bacterioplankton communities in the coastal waters of Kuwait, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.09.016

A. Almutairi / Marine Pollution Bulletin xxx (2015) xxx–xxx

Fig. 5. Relative abundance and taxonomic classification of study sites 16S rRNA bacterial clone library sequences at the family level. The taxonomic identities of checked sequences were verified using the ribosomal Database Project classifier.

subphyla Alphaproteobacteria and Gammaproteobacteria. The sequences belonging to Bacteroidetes clustered with Flavobacteriia, Sphingobacteriia, and the unclassified Bacteroidetes-related sequences of the environmental clones and isolates. 4. Discussion The Kuwaiti marine environment has been and is still one of the least investigated regions for marine microbial diversity. In this study, the DNA fingerprinting and clone library results demonstrate that the bacterioplankton composition and structure are highly dynamic within the small area of the coastal waters of Kuwait. Alphaproteobacteria were the most abundant class in the Kuwait Bay and Al-Sabbiya sites, followed by Gammaproteobacteria. However, in the Al-Khairan area, Bacteroidetes and Gammaproteobacteria were more prevalent. The high abundance of Gammaproteobacteria in Kuwait bay and AlKhairan and the predominance of Bacteroidetes in Al-Khairan indicate the impact of anthropogenic activities (industrial and domestic effluents) and sewage discharge in increasing nutrient input and altering the trophic status of these areas and subsequently modulating the composition of their bacterioplankton community. The results show that the bacterioplankton biodiversity in the coastal Kuwaiti waters exhibited distinct temporal and spatial patterns. The obtained distribution and abundance patterns were mainly influenced by changes in temperature, nutrient input and, to some extent, by the nature of the spatial location. The temperature changes appeared to be one of the plausible drivers of the seasonal changes in the composition of the bacterioplankton community. Increased temperature is known to affect the growth and drive compositional shifts in marine microbial communities (Shiah and Ducklow, 1994; Műren et al., 2005).

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The increase in the chlorophyll-a concentrations is usually associated with peaks in phytoplankton biomass and a decrease in bacterial abundance (Li et al., 1993; Fuhrman et al., 2006). However, the results show that the chlorophyll-a levels were more or less similar throughout the year at all sites (Table 2), suggesting that the seasonal changes in the chlorophyll-a levels are not sufficient to drive the diversity of the bacterial community. Thus, the shift of the bacterioplankton communities in all sites is more likely related to the changes in temperature, and this signifies the importance of temperature as a critical environmental factor regulating the abundance of bacterioplankton in the marine environment (Andersson et al, 2010; Gilbert et al, 2012). Kuwait's waters are usually oxygen-rich (Al-Yamani et al., 2004); however, the seasonal increase in water temperature during the summer caused an observed decrease in the dissolved oxygen levels, reaching as low as 4.98 mg/L at the Al-Khairan site in summer (Table 2). This effect is typically associated with a decrease in the primary productivity level and an increase in organic matter degradation, which is associated with high levels of ammonia production (Table 2). The dissolved oxygen levels of many coastal ecosystems are adversely affected by eutrophication and this phenomenon has been found to be closely linked to anthropogenic nutrient enrichment (Kemp et al., 2005). The increased nutrient input creates additional eutrophication conditions that support microbial growth and respiration, which decreases the dissolved oxygen levels in the bottom waters (Cloern, 2001; Kemp et al., 2005) and provides a massive amount of substrates for the growth and abundance of specialized bacterial groups in these waters. In the marine environment, the dissolved organic carbon concentration has a significant impact on microbial community growth and composition (Jones et al., 2009). The Kuwait Bay and Al-Khairan areas are under the influence of treated and raw sewage discharge, as well as other anthropogenic activities, and this affect the levels of organic carbon received by these areas. The total organic carbon levels were variable between the two sites and also varied with changes in temperature. The total organic carbon levels were high in spring, but increased with the increase in summer temperature. However, the average total organic carbon and polyaromatic hydrocarbon concentrations were higher around the Al-Khairan area compared to those at Kuwait Bay. Nitrogen and phosphorus are important for the growth of bacterioplankton. The co-limitation of nitrogen and phosphorus in the bacterial community could potentially impact the cycling of organic matter by bacterioplankton (Azam, 1998). Anthropogenic activities and treated and raw sewage discharge into the marine environment increase the nitrogen levels in water. The addition of inorganic nutrients, such as nitrogen, either alone or in combination with phosphorus or organic carbon, can stimulate the growth of bacterioplankton (Toolan et al., 1991; Elser et al., 1995). In the open ocean, phosphorus availability may limit bacterioplankton growth (Cotner et al., 1997). However, in the study, the recorded phosphate concentrations indicate that phosphate does not limit bacterioplankton growth in Kuwaiti waters. Although the phosphate concentrations varied between sites, they were higher in Al-Khairan. In both sites, the phosphate levels increased in summer, likely because of the decomposition of organic matter, which is usually linked to oxygen consumption and the regeneration of inorganic nutrients, such as phosphate (PO− 4 ), carbon dioxide (CO2) and ammonium (NH+ 4 ), that are necessary to sustain the continued primary production (Ward et al., 2009). Ammonium (NH+ 4 ) is the first product of organic matter decomposition and deamination, which rapidly diffuses into the water column (Diaz and Rosenberg, 2008). A wide range of ammonium concentrations was reported in Kuwait Bay and Al-Khairan, but it reached its maximum values in the summer. The ammonium concentrations in Kuwait Bay tend to increase with the increase in total organic matter concentrations. Generally, the oxidation of ammonium by bacterioplankton is the first rate-limiting step in nitrification and results in the formation of nitrite and nitrate, which will be subsequently reduced to atmospheric nitrogen (Zehr and Kudela, 2011). Hence, any decrease in the

Please cite this article as: Almutairi, A., Spatial–temporal variations and diversity of the bacterioplankton communities in the coastal waters of Kuwait, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.09.016

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Fig. 6. Phylogenetic tree based on the analysis of representative 16S rRNA sequences obtained from the three study sites bacterial clone libraries. The tree was constructed using the neighbor joining method in MEGA6. Bootstrap analysis was conducted using 2000 replicates. Bootstrap values are shown for branches with N50% bootstrap support. The16Sr DNA sequence of Methanococcoides burtonii was used as outgroup.

Please cite this article as: Almutairi, A., Spatial–temporal variations and diversity of the bacterioplankton communities in the coastal waters of Kuwait, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.09.016

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ammonium concentrations should be followed by an increase in the levels of nitrite and nitrate. Over the study period, the nitrite and nitrate levels fluctuated remarkably in Kuwait Bay and Al-Khairan, but Kuwait Bay area experienced a sharp decrease in the ammonium concentrations and an increase in the nitrate and nitrite concentrations in autumn, whereas in Al-Khairan, this shift occurred during the summer season. This indicates that the nitrifying bacterioplankton in Kuwait Bay flourish during the colder seasons and are more abundant in Al-Khairan during the warmer seasons. Interestingly, the peaks of the nitrite and nitrate concentrations increased with the increase in the levels of total organic carbon; however, the mean concentration of ammonium, nitrite and nitrate was higher in the Kuwait Bay area. Ammonia oxidation is usually performed by Crenarchaea, certain groups within the Betaproteobacteria and by the metabolically versatile Gammaproteobacteria (Diaz and Rosenberg, 2008), which explains the prevalence of Gammaproteobacteria in the Kuwait Bay area. The DGGE-based approach applied in the study was an effective method of monitoring the structure and dynamics of the bacterioplankton communities at the surveyed sites. It revealed that the major shifts in the community occurred on a seasonal basis and that higher marine bacterial diversity was observed in the summer. The cluster analysis of the DGGE fingerprint showed that the bacterial community-level changes in sequenced DGGE bands were better explained by temporal patterns than by spatial patterns (Fig. 2). The knowledge of these patterns provides insight into the bacterioplankton abundance and distribution in the Kuwaiti marine ecosystem. The bacterial diversity of the constructed clone libraries exhibited a spatial gradient of increasing diversity from north to south along the Kuwaiti coast, with the highest diversity at Al-Khairan (Table 3). Although Al-Sabbiya receives river discharge, which is expected to be associated with more eutrophic conditions and eutrophication-associated organisms, the bacterioplankton richness and diversity of Al-Sabbiya was the lowest of the three sites. Typical marine phyla, such as Proteobacteria and Bacteroidetes, were predominant in the Kuwaiti marine environment. These bacterial groups were widely distributed at the surveyed sites. Marine microbial diversity studies have shown that the most abundant class was the Alphaproteobacteria, which was corroborated in this study. Alphaproteobacteria was the more abundant class at all study sites and was the most abundant at Al-Sabbiya. Within this class, the most recorded sequences were members of the orders Rhodobacteriales and Rhodospirillales. The sequences belonging to the order Caulobacterales were found only in Al-Sabbiya and Al-Khairan, and not in Kuwait Bay. The sequencing of the prevalent DGGE bands revealed that the predominant family of the order Rhodobacteriales was the physiologically highly diverse family Rhodobacteraceae, which is one of the major groups of marine bacteria that usually comprise up to 20% of the coastal bacterioplankton communities (Buchan et al., 2005). The predominant member found of this family was the genus Roseovarius within the Roseobacter clade, along with several uncultured members of this clade. The appearance of this band at all sites, with no temporal and spatial preference, suggests that the Roseobacter clade was present throughout the year and that this clade is potentially more resilient than other bacterial groups to the changing environmental conditions of Kuwaiti waters. Previous studies have shown that some members of the Roseobacter clade exhibit a systematic global distribution in the surface water of the temperate to polar oceans of both hemispheres (Selje et al., 2004; Buchan et al., 2005). The adaptation of members of the Roseobacter clade to varying environmental conditions reflects their high genetic and metabolic activity (Buchan et al., 2005; Newton et al., 2010). The Roseobacter clade is of major importance for the cycling of carbon in the marine environment, and many members of the Roseobacter clade contain metabolic features that allow them to thrive in various marine environments (Wagner-Dobler and Biebl, 2006; Newton et al., 2010).

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Generally, bacteria belonging to the Rhodobacteraceae family follow the seasonal development of primary producers in the marine environment. In particular, members of the Roseobacter clade, which is exclusive to marine or hypersaline environments (Buchan et al., 2005), tend to peak when levels of primary productivity are high (Brinkhoff et al., 2008; Gilbert et al., 2012). Studies have shown that in a system that is constantly provided with nutrients, the members of the Roseobacter clade maintained a consistently high population throughout the year (Alonso-Gutiérrez et al., 2009); thus, their prevalence in Kuwait waters strongly confirms the high primary productivity of these waters. Members of the SAR11 cluster were found at all study sites, which is not surprising as the SAR11 clade is a dominant component of the marine bacteria in surface ocean waters (Morris et al., 2002; Giovannoni and Rappé, 2000). The growth of the SAR11 clade may benefit from the higher light levels at the ocean's surface and may depend on the nature of the organic substrate (Tada et al., 2011). The obtained SAR11 clones were closely related both to C. pelagibacter, which was isolated from the surface of a fresh water lake, and to the uncultured C. pelagibacter ubique fosmid, which was sampled at a coastal marine habitat. The sequencing of the DGGE bands also revealed that at AlKhairan, the SAR11 cluster, and C. pelagibacter ubique in particular, were prevalent, regardless of the season. Members of the SAR11 clade are usually considered oligotrophic scavengers, but they were found throughout the year in the nutrient-rich Kuwaiti waters (Table 2), indicating the extraordinarily ubiquitous distribution of the members of the SAR11 cluster along the Kuwaiti coast. The second most dominant phylum in all study sites was Gammaproteobacteria, and the sequenced clones were very versatile. Members of the order Altermonadales were predominant at all study sites. The obtained sequences were affiliated with the sequences retrieved from different environments, such as oceanic waters, coastal waters, hydrothermal sediments, corals, sponges, and salt marshes, indicating the high abundance of this phylum in various marine habitats. Interestingly, a number of the obtained Gammaproteobacteria sequences were closely related to bacterial species that display versatile metabolic activities, such as the degradation of crude oil, dibenzofuran, polysaccharide, naphthalene, phenanthrene, and carbazole, as well as the assimilation of nitrate. Those clones were found in Kuwait Bay and Al-Khairan, but not at Al-Sabbiya. Studies have shown that many members of Gammaproteobacteria are opportunistic heterotrophs that specialize in the colonization of organic aggregates or other nutrient-rich microniches (Pernthaler and Amann, 2005). Therefore, the high level of anthropogenic activities and petroleum-related industries in the vicinity of the Kuwait Bay and Al-Khairan sites may lead to the discharge of petroleum hydrocarbons into the coastal environment. Moreover, the presence of similar petroleum hydrocarbon levels throughout the study period at Kuwait Bay area and around Al-Khairan area (Table 2) clearly encouraged the growth of those bacterial groups. The most prevalent member of Gammaproteobacteria at Kuwait Bay and Al-Khairan was the globally distributed genus Alteromonas. Usually, members of Alteromonas dominate heterotrophic blooms, thriving on available organic nutrients (McCarren et al., 2010), and the prevalence of these bacteria at Kuwait Bay and Al-Khairan demonstrates the copiotrophic nature of these bacteria. Previous studies have shown that many Alteromonas species play important roles in hydrocarbon degradation in the marine environment, and the isolation of hydrocarbondegrading Alteromonas from Kuwaiti corals provided evidence of such activity (Al-Dahash and Mahmoud, 2013). Furthermore, the presence of clones of the hydrocarbon-oxidizing genus Pseudoaleteromonas (Melcher et al., 2002; King et al., 2012) in the Kuwait Bay and AlKhairan sites also support the role of hydrocarbon availability in structuring the prevalent members of Gammaproteobacteria. The phylum Bacteroidetes is a widely distributed group of chemoheterotrophic bacteria that live in marine habitats (Cottrell and Kirchman, 2000). The Bacteroidetes were well represented at all sites

Please cite this article as: Almutairi, A., Spatial–temporal variations and diversity of the bacterioplankton communities in the coastal waters of Kuwait, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.09.016

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and were the predominant group at Al-Khairan. The obtained sequences were affiliated with the Flavobacteriia and Sphingobacteriia sequences from freshwater and marine habitats in other geographic locations. However, several of these sequences were related to unclassified Bacteroidetes. The sequencing of DGGE bands showed that Bacteroidetes sequences were obtained from the Al-Khairan summer samples (i.e., June and August), suggesting that these sequences are summer-associated species that adapt to the high summer temperatures in Kuwait. Bacteroidetes usually dominate during bloom periods, when high nutrient input could favor these organisms (Pinhassi and Hagstrom, 2000; Alonso and Pernthaler, 2006). The southern coast of Kuwait is characterized by beds of benthic Sargassum, and these algae grow best at lower temperatures; however, increased water temperatures cause them to break free of their holdfasts and float to the surface. The disintegration of these algae provides readily available organic compounds to support the growth of the Bacteroidetes in this area. The Al-Khairan area also experiences high levels of anthropogenic contamination and crude oil spillage from industrial and petrolrelated transport. The area received large quantities of untreated sewage discharge as a result of the major pumping failure at the Mishref sewage station. Hence, the coastal waters in the south were contaminated with higher concentrations of organic matter (Table 2) that favored the growth of heterotrophic bacteria and promoted the abundance of Bacteroidetes. Previous studies demonstrated that Bacteroidetes prefer high molecular weight dissolved organic compounds (Cottrell and Kirchman, 2000), and their abundance increases in eutrophic waters (Lydell et al., 2004). Interestingly, at the Al-Khairan area, the levels of chlorophyll-a and phosphate increased in summer, which should lower the bacterial growth potential to some extent. However, the presence of elevated nitrate levels indicates that nitrate was not completely used by the primary producers, and the available organic matter supply allowed the growth of the present bacterial community. Moreover, the anticlockwise water circulation pattern found in the Kuwaiti coastal water and the Arabian Gulf in general may have helped distribute the raw sewage around the Arabian Gulf and transport Bacteroidetes to the northern site, including Al-Sabbiya. 5. Conclusions This study is the first to provide a basic biogeography of the bacterioplankton diversity within the small area of the Kuwaiti coastal waters. The study presents primary evidence for the temporal and spatial patterns occurring in the bacterioplankton community and showed that they are largely influenced by natural environmental factors and nutrient availability, mainly from the sewage pollutants already known for the marine environment of Kuwait. The results show that Proteobacteria and Bacteroidetes dominated Kuwaiti waters, and, on a seasonal scale, Alphaproteobacteria was the most abundant class in these waters. Members of the Roseobacter clade, which were found to be more resilient and maintain consistent growth in the trophic conditions of Kuwait waters, were mostly independent of the environmental conditions, suggesting that the metabolic features of the members of the Roseobacter clade supported their growth in the nutrient-rich Kuwaiti waters. The SAR11 clade was also found to be ubiquitous in Kuwaiti waters, regardless of the nutrient concentration and the seasonal changes in temperature. It is possible that the SAR11 clade members found in local waters may belong to lineages that are adapted to more eutrophic conditions. The results from the clone library analysis revealed that despite the trophic status present in the local waters, the studied sites differed markedly in their dominant bacterial phylotypes. The spatial heterogeneity of the bacterioplankton distribution in the surveyed sites gave evidence of the immense anthropogenic pressure on these sites and the ways in which the discharge of contaminants from different origins can shape the microbial diversity present. Sewage disposal and the organic matter supply gave rise to the high diversity of Gammaproteobacteria and Bacteroidetes in Kuwait Bay and Al-

Khairan. The presence of clones affiliated with bacterial pathogen, such as Coxiella, which is associated with sewage water treatment plants, highlight the health risks of sewage discharge in the marine environment. This study provides useful insights into the bacterioplankton communities in Kuwaiti coastal waters and the influence of nutrient input disturbances, mainly from anthropogenic activities, on the dynamics of microbial populations within Kuwaiti waters. The results clearly show that the spatial distances did generate a considerable difference in the bacterioplankton diversity and abundance found within the small area of Kuwaiti seawater, indicating the vulnerability of the marine ecosystem of Kuwait and the immense anthropogenic pressure on this system. These results highlight the need for further studies to better understand the overall dynamics and abundance of the bacterioplankton community assemblages and the exact processes that control their distribution and productivity in the marine environment of Kuwait. Acknowledgments The excellent assistance of Parvathy Anitha is gratefully acknowledged. The help of my excellent student Shahad Al-Roumi during clone library preparation is highly appreciated. The author wishes to thank the SAF Unit and the Biotechnology Center of Kuwait University for the support provided through projects GS 02/01 and GS 01/02. Thanks are also due to the Kuwait Environmental Public authority (KEPA) for providing annual statistical bulletin of the environment for 2010 and 2011. References Al-Dahash, L.M., Mahmoud, H., 2013. Harboring oil-degrading bacteria: a potential mechanism of adaptation and survival in corals inhabiting oil-contaminated reefs. Mar. Pollut. Bull. 72, 364–374. Al-Enezi, E., Al-Dousari, A., Al-Shammari, F., 2014. Modeling adsorption of inorganic phosphorus on dust fallout in Kuwait Bay. J. Eng. Res. 2, 1–14. Al-Ghadban, A.N., El-Sammak, A., 2005. Sources, distribution and composition of the suspended sediments, Kuwait Bay, Northern Arabian Gulf. J. Arid Environ. 60, 647–661. Al-Ghadban, A.N., Al-Majed, N., Al-Muzaini, S., 2002. The state of marine pollution in Kuwait: Northern Arabian Gulf. Technology 8, 7–26. Al-Muzaini, S., 2002. Sewage discharge impact on the development of the Shuwaikh area. Tech. J. 8, 51–54. Al-Nafisi, R.S., Al-Ghadban, A., Gharib, I., Bhat, N., 2009. Positive impacts of mangrove plantations on Kuwait's coastal environment. Eur. J. Sci. Res. 26, 510–521. Alonso, C., Pernthaler, J., 2006. Roseobacter and SAR11 dominate microbial glucose uptake in coastal North Sea waters. Environ. Microbiol. 8, 2022–2030. Alonso-Gutiérrez, J., Lekunberri, I., Teira, E., Gasol, J.M., Figueras, A., Novoa, B., 2009. Bacterioplankton composition of the coastal upwelling system of ‘Ría deVigo’, NW Spain. FEMS Microbiol. Ecol. 70, 493–505. Al-Rifaie, K., Al-Yamani, F., Polikarpov, I., 2008. First study of the bacterioplankton distribution in the Northwestern Arabian Gulf. Mar. Ecol. J. 574, 43–48. Al-Yamani, F., 2001. A Strategic Plan for Sustainable Utilization of Kuwait's Marine Environment. Kuwait Foundation for Advancement of Sciences, p. 140. Al-Yamani, F., Durvasula, R., Ismail, W., 1997. Dynamic Oceanography of the Northwestern Waters of the Arabian Gulf: Ecological Significance of the Marine Food web/ Rep. No. KISR 5173. Kuwait Institute for Scientific Research, Kuwait, p. 238. Al-Yamani, F.Y., Bishop, J., Ramadhan, E., Al-Husaini, M., Al-Ghadban, A., 2004. Oceanographic Atlas of Kuwait's Waters. Kuwait. Institute for Scientific Research, Kuwait. Al-Yamani, F., Subba Rao, D.V., Mharzi, A., Ismail, W., Al-Rifaie, K., 2006. Primary production off Kuwait, an arid zone environment, Arabian Gulf. Int. J. Oceans Oceanogr. 1, 67–85. Andersson, A.F., Riemann, L., Bertilsson, S., 2010. Pyrosequencing reveals contrasting seasonal dynamics of taxa within Baltic Sea bacterioplankton communities. ISME J. 4, 171–181. Azam, F., 1998. Microbial control of oceanic carbon flux: the plot thickens. Science 280, 694–696. Brinkhoff, T., Giebel, H.A., Simon, M., 2008. Diversity, ecology, and genomics of the Roseobacter clade: a short overview. Arch. Microbiol. 189, 531–539. Buchan, A., González, J.M., Moran, M.A., 2005. Overview of the marine Roseobacter lineage. Appl. Environ. Microbiol. 71, 5665–5677. Buerger, S., Spoering, A., Gavrish, E., Leslin, C., Ling, L., Epstein, S.S., 2012. Microbial scout hypothesis and microbial discovery. Appl. Environ. Microbiol. 78, 3229–3233. Cloern, J.E., 2001. Our evolving conceptual model of the coastal eutrophication problem. Mar. Ecol. Prog. Ser. 210, 223–253. Colwell, R.K., Coddington, J.A., 1994. Estimating terrestrial biodiversity through extrapolation. Philos. Trans. R. Soc. Lond. B 345, 101–118.

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Please cite this article as: Almutairi, A., Spatial–temporal variations and diversity of the bacterioplankton communities in the coastal waters of Kuwait, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.09.016