Spatial variation of bacterial community composition ...

GENE-40917; No. of pages: 8; 4C: 3, 4, 5 Gene xxx (2015) xxx–xxx

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Research paper

Spatial variation of bacterial community composition at the expiry of spring phytoplankton bloom in Sendai Bay, Japan Tomoko Sakami a,⁎, Tsuyoshi Watanabe a,b, Shigeho Kakehi a, Yukiko Taniuchi c, Akira Kuwata a a b c

Tohoku National Fisheries Research Institute, Fisheries Research Agency, 3-27-5 Shinhama, Shiogama, Miyagi 985-0001, Japan CREST, JST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan Hokkaido National Fisheries Research Institute, Fisheries Research Agency, 116 Katsurakoi, Kushiro, Hokkaido 085-0802, Japan

a r t i c l e

i n f o

Available online xxxx Keywords: Bacterial diversity Pyrotag sequencing Phytoplankton sedimentation Hydrographic events

a b s t r a c t In order to characterize how bacterial communities are propagated over spatial scales in a coastal area, the bacterial community composition was examined along with a transect line set in a bay at an expiry of spring phytoplankton bloom. Four distinctive bacterial communities were found within the bay by a fingerprinting method of 16S rRNA gene amplicons. The most widely distributed one was distributed in the surface and middle layers at whole area of the bay. The water was characterized by low inorganic nutrients concentration and high bacterial abundance, suggesting that the bacterial community had been developed in the bloom. Pyrosequencing analyses of the gene amplicons indicated that Rhodobacteriaceae and Flavobacteriaceae were abundant in the bacterial community, though the most abundant bacterial taxon was SAR11. The second group was distributed in the bottom water at the coastal side of the bay where considerably high Chl. a concentration was observed, probably because of the sedimentation of phytoplankton bloom. The community diversity was high and Alteromonadaceae, Saprospiraceae, and some families of Actinobacter existed more in this community than the others. The third group was distributed in the deep water near the border with the outside of the bay. The ratio of SAR11 was the highest in this community; besides, Burkholderianceae and Rhodospilliraceae existed in relatively high abundances. Another bacterial community having intermediate characters was observed in the middle to bottom layers around a central part of the bay where vertical water mixing was observed. These findings suggest that spatially different bacterial communities were formed under the influences of phytoplankton bloom and/or hydrographic events such as oceanic seawater intrusion of the bay. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction In coastal areas, biological productivities are often high because of terrestrial nutrient inputs and various marine organisms are brought up there. However, they are easily disturbed by over-loading of nutrients discharged from city area and/or agricultural fields. Artificial architectures such as harbor and flood walls also give substantial effects on coastal environments. To preserve a proper environmental condition, biological diversity should be considered. Bacteria have principle roles in organic matter degradation and nutrients regeneration in marine ecosystems and it can respond directly and quickly to the change of allochthonous nutrient input (Murrell et al., 1999; Paerl et al., 2003).

Abbreviations: rRNA, ribosomal RNA; Chl. a, chlorophyll a; T-RFLP, terminal restriction enzyme digestion fragment length polymorphism; DO, dissolved oxygen; SCM, subsurface chlorophyll maximum; DMF, N, N′-dimethylformamide; MID, molecular identifier; OTU, operational taxonomic unit; PCA, principal component analysis. ⁎ Corresponding author. E-mail address: [email protected] (T. Sakami).

Therefore, bacterial community composition may become a good indicator to assess environmental changes in a coastal marine ecosystem. Bacterial community diversity is generally examined by using ribosomal RNA genes because most bacteria living in natural environments are unculturable. At coastal areas, it has been revealed that bacterial community composition changes depends on environmental factors such as temperature, salinity, and nutrient concentrations (Kirchman et al., 2005; Fuhrman and Hagström, 2008; Sakami, 2008; Fortunato et al., 2013; Yeo et al., 2013). Phytoplankton blooms and/or eutrophication of water also give effects on bacterial communities (Pinhassi et al., 2004; Tada et al., 2011; Liu et al., 2013). However, it has been difficult to know the exact bacterial community composition including minor species because its diversity is often too large to examine by using ordinal low-resolution methods such as a combination of clone library and sanger-sequencing or some finger printing methods (Gobet et al., 2012). Moreover, environment conditions vary so complicatedly at a coastal area, details of bacterial community variation still remain unknown. Recently developments of pyrosequencing machines and bioinformatics allow us to analyses a massive sequence data. Using these techniques, bacterial community dynamics have been revealed in

http://dx.doi.org/10.1016/j.gene.2015.10.011 0378-1119/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: Sakami, T., et al., Spatial variation of bacterial community composition at the expiry of spring phytoplankton bloom in Sendai Bay, Japan, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.10.011

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T. Sakami et al. / Gene xxx (2015) xxx–xxx

relation with the environmental conditions at various coastal environments (Campbell and Kirchman, 2013; Teeling et al., 2012; Wemheuer et al., 2014; Fortunato et al., 2013). Phytoplankton bloom is one of the most important biological events in marine water, where there are strong interactions between phytoplankton and bacteria. Some bacteria produce chemical substances that promote phytoplankton growth, such as vitamin B12 (Starr et al., 1957) or indole-3-acetic acid that acts as a hormone to a diatom species (Amin et al., 2012, 2015). On the other hand, many kinds of bacteria inhibit the growth of or kill phytoplankton in seawater (Park et al., 2010). The abundance of phytoplankton growth-inhibiting bacteria increases correspondingly with the level of chlorophyll a in seawater, suggesting that these bacteria play a significant role in regulating phytoplankton abundance in coastal marine environments (Inaba et al., 2013). Changes of bacterial community composition have been well studied in relation to a phytoplankton bloom. The community composition and dominant species changed accompany with the bloom development as well as bacterial abundance, growth rate, and some enzymatic activities (Pinhassi et al., 2004). The active bacterial species were also different between the inside and the outside of the blooming area (Tada et al., 2011). However, there are few studies about a spatial distribution of such a unique bacterial community in relation with the bloom occurrence. Moreover, bacterial community composition may be different depends on the water depth in a shallow coastal area, because a bacterial community in water may have an interaction with that in the beneath sediment (Yeo et al., 2013; Hamdan et al., 2013). To elucidate bacterial community variation at a coastal area, spatial variations should be clarified as well as temporal variations. Sendai Bay locates at the north east part of Japan and it has a simple semicircle form with a wide open facing to the Pacific. Some rivers are discharged into the bay to give considerable effects on hydrographic properties, as well as an oceanic water flow into the bay (Kudo, 1971; Kakehi et al., 2012). However, a uniform water mass is formed within the bay in winter, because the reduction of river water discharge and cooling of water surface cause a strong water mixing. Consequently a phytoplankton bloom, which is constituted mainly by diatoms, is developed in the nutrient rich water mass in spring. The purpose of this study is to elucidate the spatial variation of bacterial community composition relating to the phytoplankton bloom. We examined bacterial community composition along with a transect line in the Sendai Bay at the expiry of spring bloom. A terminal restriction enzyme digestion fragment length polymorphism (T-RFLP) method was used to know the

distribution of distinctive bacterial communities, and an ampliconpyrosequencing analysis was done for some representative samples to know details of bacterial community variations. 2. Materials and methods 2.1. Sample collection and DNA extraction The C line (Fig. 1) which ran from stations C1 to C13 was a crossshore line located off the Abukuma River. Hydrodynamic profiles of temperature, salinity, dissolved oxygen (DO) were measured by aqua quality sensor AAQ (AAQ-1186, JFE Advantech). Water samples were taken at depths 1 (surface layer), 10, 30 and 50 m (middle layer), and at 0.5 m above the bottom (bottom layer). Water samples were also collected at the sub-surface Chlorophyll maximum (SCM) which was determined from the profile of Chl. a measured by the fluorescence sensor of the AAQ. The salinities of the AAQ were calibrated with the salinity measured by the salinometer (AUTOSAL 8400B, Guildline Instruments Ltd). Water samples for Chl. a analysis were filtered through a 25 mm Whatman GF/F glass fiber filter and preserved in DMF (N, N′dimethylformamide). Fluorometric measurements of Chl. a were performed using a Turner Designs fluorometer (10-AU, Turner Designs). Values of fluorescence sensor of the AAQ were calibrated with fluorometrically measured Chl. a concentrations. Inorganic nutrient concentrations were determined using an Auto-Analyzer (QuAAtro, BL-TEC). Bacterial abundance was determined using a fluorescent microscopy (Porter and Feig, 1980). Major phytoplankton species were identified from seawater samples fixed with acid Lugol solution (4% final concentration) under light microscopy (Tomas, 1997). DNA water samples were collected at stations C1–5, 7, 10, 12, and 13. Water samples were cooled immediately after collection and kept in dark. Waters of 300 to 500 mL were filtered through a membrane filter of normal pore size 0.22 μm (GS, Millipore) within 10 h after sample collection. Filters were stored frozen at −80 °C until processing. To avoid an inhibitive effect of high concentration of suspended matters in the bottom water, DNA was extracted using a FAST DNA-SPIN kit for soil (MP. Biomedicals) in accordance with the manufacturer's instructions. 2.2. Terminal restriction enzyme fragment length polymorphisms (T-RFLP) The bacterial community structure in the water was examined using T-RFLP of PCR-amplified 16S rRNA gene fragments. PCR was conducted

Fig. 1. Locations of observation stations and bathymetry in Sendai Bay.



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with 6-carboxyfluorescein-labeled 27F and 519R primers with an initial denaturation at 94 °C for 3 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min; final elongation was carried out for 7 min at 72 °C (Danovaro et al., 2006). Triplicate PCR products were combined and purified using the NucleoSpin Gel and PCR Clean-up kit (Takara Bio). Approximately 100 ng of each purified amplicon was subsequently digested with Hae III (Takara Bio) at 37 °C for 6 h, and approximately 10 ng of the digested sample was used for electrophoresis on a capillary DNA sequencer (ABI 3100, Applied Biosystems). Peak scanner 2 software (Applied Biosystems) was used to estimate the size and relative abundance of the resulting terminal restriction fragments (T-RFs). The top 30 peaks were determined in each sample and fragment length were rounded to the nearest integer values, aligned, and manually corrected for likely errors in peak determination due to such factors as instruments drift, etc. 2.3. Multitag pyrosequence analysis First PCR was conducted using 27Fmod and 338R primers (Kim et al., 2013) modified by adding 33 and 34 bp nucleotide respectively to add molecular identifier (MID) tags later, with an initial denaturation at 95 °C for 2 min, followed by 20 cycles of denaturation at 95 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 30 s; final elongation was carried out for 1 min at 72 °C. Product was visualized on an agarose gel with SYBR® Safe (Life technologies), excised and purified from the gel and quantified by using a QuantiFluor™ (Promega). Approximately 10 ng of the first PCR product was used as template for the second PCR conducted to add MID tags using a forward (59 bp) and reverse (54 bp) primer set with an initial denaturation at 98 °C for 30 s, followed by 12 cycles of denaturation at 98 °C for 10 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s; final elongation was carried out for 1 min at 72 °C. The PCR programs were determined in accordance with the sequencing machine operating company's instruction (FASMAC Co. Ltd). Sequencing was performed on the Illumina MiSeq by 300 bp paired end run. Data were analyzed by sickle tools (v 1.200) for quality filtering and FLASH (v 1.2.7) for paring end and removing primers and MID tag sequences. Chimera was removed on the RDP's Pipeline (Edgar et al., 2011). Sequences were classified and clustered into operational taxonomic units (OUTs) in MOTHUR (Schloss et al., 2009) according to the MiSeq standard operation procedure (SOP) (Kozich et al., 2013). Sequences are available in the DDBJ Sequence Read Archive database, accession number DRA002564.

Fig. 2. Contour plots of (a) temperature, (b) salinity and (c) Chl.a at C-line in Fig. 1. Circles in (c) indicate depths which water samples for bacterial community analysis were taken.

less than 0.5 μg/L were observed at the surface layer of the whole area and the bottom layer near the border with the outside of the bay (C12, C13). Phytoplankton was dominated by diatoms, which comprised Skeletonema costatum (28%), Thalassionema nitzschioides (8%),

2.4. Statistics Principal component analysis (PCA) was performed using R (v 3.0.2) to infer relationships of the T-RFLP profiles. Relationships between the first or the second PCA score and environmental variables were examined by person correlation coefficients. Relative abundance of bacterial families or major OTUs was compared among the bacterial community groups determined by T-RFLP profiles using student t-test. 3. Results Water stratification was weakly observed from the hydrographic properties of temperature and salinity at the sampling time, accordingly an estuary circulation was driven and oceanic seawater with low temperature and high salinity was intruded into the bay through the bottom layer (Fig. 2). At St. C7, the stratification was destroyed and the high temperature and salinity was observed through the water column. Chl. a concentration was high at the middle layer of coastal (C1) and central (C6–C8) parts of the bay. In addition, high Chl. a concentration more than 5 μg/L was also observed at the bottom layer at coastal side (C1–C5) of the bay. On the other hand, low Chl. a concentrations

Fig. 3. Contour plots of (a) nitrate concentration, and (b) bacterial abundance at C-line in Fig. 1.


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Chaetoceros debilis (8%), Pseudo-nitzschia seriata (7%), Chaetoceros compressus (7%), and Asterionellopsis glacialis (6%). Inorganic nutrient concentrations were low at the surface and middle layers, but high at the bottom layer on the whole. The nitrate concentration was remarkably high at the bottom water of St. C13 (Fig. 3), as well as those of nitrite and phosphate. On the other hand, the ammonia concentrations were high at St. C1 (3.4 μM) and St. C7 (2.3 μM), but low at St. C5 (0.5 μM) and St. C13 (0.7 μM). Bacterial abundance was higher at the surface than the middle and bottom waters. The bacterial communities in the Sendai Bay were grouped into four types by the PCA analysis of the T-RFLP patterns (Fig. 4). Bacterial communities having positive values in the second principle component (PC2) score were distributed at the surface and middle layers throughout of the bay (Group I). Those having negative values in PC2 score were distributed in the bottom layer of the bay and recognized in three different groups. The first one (Group II) have the small values in PC1 score. They distributed at the bottom layer at the coastal side (C1–C5) of the bay. The second one (Group IV) have the large values in PC1 score and distributed at the deep waters near the border with the outside of the bay (C12, 13). Between Group II and Group IV, the third one (Group III) was distributed at stations C7–C10 where the water stratification collapsed. The distribution pattern of Group II, III, and IV was corresponded to that of Chl. a concentration in the bottom waters. Inorganic nutrient concentrations and bacterial abundance were negatively correlated with the PC2 score, as well as temperature, salinity, and water depth (Table 1). On the other hand, Chl. a concentration was negatively correlated with the PC1 score. Sixteen samples were selected for 16S rRNA gene amplicon pyrosequencing based on the result of T-RFLP analysis (Table 2). Finally 115,089 (1067–14,904) sequences were obtained after some quality filterings and they were clustered in 8398 OTUs at 97% similarity. Rarefaction curve analysis indicated that 80–93% of the community members were covered by these sequences. The coverage ratio was low and the inverse of Simpson index was high at Group II, indicating that this bacterial community was more diverse than the others. Proteobacteria was the most abundant bacterial phylum (68–85%, 77% on average) in all of the examined samples (Fig. 5). In the Proteobacteria, Alphaproteobacteria was existed at a range from 19 to 51% and less abundant in Group II. Gammaproteobacteria (18–53%) was less abundant in Group IV. Betaproteobacteria (7–26%) was remarkably abundant

Table 1 Correlation coefficients between the first two PCA scores and environmental variables.

Depth Temperature Salinity DO (mg/L) NO3–N NO2–N PO4–P SiO2–Si NH4–N Chl. a Bacterial abundance

PC1

PC2

0.24 −0.02 0.30 0.27 0.35 −0.12 −0.06 0.21 −0.18 −0.58 0.01

−0.86 0.59 −0.57 0.26 −0.80 −0.64 −0.75 −0.78 −0.52 −0.33 0.77

Bold types indicate the significant values at p b 0.05.

in the bottom water at St. C13 of Group IV. Deltaproteobacteria and Epsilonproteobacteria were more abundant in Group II than the other groups. The next abundant phylum was Bacteroides (7–26%) and it was less abundant in Group III and IV. Actinobacteria was observed more abundantly in Group II at a range from 2 to 3%. Major bacterial families detected from the Sendai Bay were SAR11 (8–33%, 20% on average), Rhodobacteriaceae (10–17%, 14% on average), and Flavobacteriaceae (6–12%, 9% on average) (Table 3). The abundances of other families were less than 2% on average. Rhodobacteriaceae and Flavobacteriaceae were abundant in Group I. In Group II, SAR11 was less abundant and some families appeared simultaneously; Alteromonas and Saprospiraceae appeared at relatively high abundances as much as 5%. Iamiacea and Acidimicrobidae-incertae-sed, which belongs to Actinobacteria, also appeared much. Colwelliaceae, Moritellaceae, Psychromonadaceae, and Verrucomicrobiaceae were also occurred abundantly in Group II, or tended to be much in Group III. Shewanellae and Vibriaceae appeared much in the bottom communities (Group II to IV). In Group IV, the abundance of SAR11 was the highest among the four groups. Burkholderiaceae and Rhodospirillaceae were also abundant in this group. Comamonadaceae appeared at an especially high ratio (0.056) in the bottom water at C13 in Group IV. The major 20 OTUs accounted for 46% of the total bacterial abundance. The most abundant OUT (OUT1 in Fig. 6) was classified into SAR11 with an average abundance of 15%. It was always the most dominant OTU in all bacterial community groups. The next abundant one (OUT3) was classified into Roseobacter with an average abundance of 6%. The relative abundances of other OTUs were less than 3% on average. Among the major 20 OTUs, four (OUT3 – 6) were classified into

Table 2 Coverage values of the community estimated from the rarefaction curve and inverted Simpson diversity indices of each samples.

Fig. 4. First 2 principal components (PC) from a principal component analysis (PCA) of environmental T-RFLP. Open circle symbols indicate samples from the surface water and solid symbols indicate those from the bottom water. Mesh densities in the circles indicate the increasing water depth.

Sample⁎

No. of seqs

Coverage

Inv-Simpson

Group I

C1-S C1-M C5-S C5-M C7-S C10-S C12-S C12-30

14,904 6848 2819 4340 10,098 1067 8696 5035

0.91 0.92 0.93 0.93 0.95 0.89 0.93 0.93

41 33 14 21 22 29 17 16

Group II

C1-B C4-B C5-B

6293 7611 4891

0.80 0.86 0.86

84 67 81

Group III

C7-B C10-B

5145 2163

0.90 0.85

31 24

Group IV

C12-50 C12-B C13-B

10,683 13,356 11,140

0.93 0.91 0.92

13 15 18

⁎ Station and depth (m), S: 1 m, M: SCM, B: bottom −0.5 m.



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Fig. 5. Relative distribution of bacterial lineages at each communities grouped by the T-RFLP profiles.

Roseobacter and five (OTU7–11) were into SAR92. As Roseobacter belongs to Rhodobacteria, this lineage seems responsible for the dominance of Rhodobacteriaceae at a family level. These OTUs tended to be abundant in Group I or I and III. Three OTUs (OTU12–14) were classified into Flavobacteria and they were abundant in Group I or I and II. The four OUTs classified into SAR11 (OTU1, 2), unclassified Gammaproteobacteria (OUT15), and Burkholderiaceae (OUT16) were abundant in Group IV. An OTU classified into SAR86 (OTU17) was abundant in Group I and IV. Besides these bacterial taxa, Oceanospiliceae, OM43 and OM182 were also found in the major OUTs.

4. Discussions The bacterial community distinguished by the T-RFLP methods was also distinctive in the pyrosequencing analysis. However, when restriction enzyme digestion fragment lengths of OTUs were estimated from their sequences, we found that multiple OTUs had the same length even when dominant OTUs were examined (data not shown). Therefore, the T-RFLP method is useful to determine the distribution pattern of bacterial community by analyzing a number of samples, but it cannot be used to determine the diversity and/or major members of the community. Nevertheless, by combining T-RFLP with pyrosequencing, we Table 3 Relative abundances of the top 20 families in the bacterial communities grouped by the T-RFLP profiles.

Rhodobacteraceae Flavobacteriaceae Alteromonadaceae Saprospiraceae Acidimicrobidae_incertae_sed Iamiaceae Colwelliaceae Moritellaceae Psychromonadaceae Verrucomicrobiaceae Shewanellaceae Vibrionaceae SAR11 Burkholderiaceae Rhodospirillaceae Comamonadaceae Oceanospirillaceae Cyanobacteria Family_I Campylobacteraceae Sphingomonadaceae (sum)

Group I

Group II

Group III

Group IV

0.170 0.124 0.013 0.004 0.002 0.010 0.002 0.000 0.000 0.001 0.000 0.000 0.213 0.003 0.002 0.011 0.017 0.002 0.001 0.000 (0.576)

0.133 0.099 0.047 0.048 0.012 0.023 0.018 0.005 0.004 0.002 0.001 0.001 0.083 0.000 0.000 0.006 0.014 0.003 0.003 0.001 (0.505)

0.152 0.055 0.029 0.010 0.006 0.017 0.018 0.006 0.004 0.002 0.006 0.006 0.163 0.004 0.005 0.007 0.018 0.003 0.002 0.001 (0.515)

0.101 0.065 0.007 0.004 0.003 0.010 0.005 0.000 0.001 0.001 0.001 0.001 0.331 0.039 0.007 0.024 0.011 0.003 0.003 0.003 (0.619)

Bold or underlined types indicate significant difference from normal italic values at p b 0.05.

determined some characteristics of bacterial communities in the Sendai Bay towards the end of spring phytoplankton bloom.

4.1. Major bacterial community in the Sendai Bay — Group I A distinctive bacterial community Group I was distributed widely in the surface and middle layers throughout the Sendai Bay. Simultaneously, Group I appeared in the water of low nutrient concentration and high bacterial abundance. Because we had observed high Chl. a concentration in the examined area on March, one month before the sampling date, it seems that the phytoplankton might have consumed most nutrients in the water. In addition, bacterial abundances usually increase at a latter period of a phytoplankton bloom (Pinhassi et al., 2004). Considering from these facts, it seems that Group I was represented a bacterial community at the expiry of the phytoplankton bloom. The major bacterial taxa in the bay were identified as SAR11 (21%), Rhodobacteriaceae (17%), and Flavobacteriaceae (12%). They appeared abundantly in Group I. These bacterial taxa are often detected from estuarine and coastal environments (Campbell and Kirchman, 2013; Yeo et al., 2013; Teeling et al., 2012). Fortunato et al. (2013) indicated that Rhodobacteriaceae species were prevalent in coastal ocean-influenced environments and Flavobacteria species were indicators for the plume environment (salinity b31) in the Columbia River estuary. Moreover, these bacterial lineages were shown to be active in the coastal water by using rRNA analysis (Campbell and Kirchman, 2013). They were also the major bacterial taxa appearing throughout a year with having high dividing activities in the Seto Inland Sea, Japan (Taniguchi and Hamasaki, 2008). Considering from these facts, it seems that these bacteria were developed in the coastal water mass which had formed within the bay during winter. Rhodobacteriaceae and Flavobacteriaceae also have been shown to be active and/or increasing their abundances in a phytoplankton bloom (González and Moran, 1997; Alonso-Sáez et al., 2007; Wemheuer et al., 2014). On the other hand, Tada et al. (2011) reported that the specific activity of Roseobacter did not differ markedly between the inside and the outside of the phytoplankton blooming area, suggesting that this kind of bacteria could keep their activities and abundance in the nutrient exhausted water. Flavobacteria also has been shown to increase its abundance when phytoplankton bloom became diminishing (Teeling et al., 2012). These facts suggest that these two bacterial lineages could keep their high abundances at the expiry of phytoplankton blooms. Among the major 20 OTUs appeared in the Sendai Bay, four were classified into Roseobacter and three were into Flavobacteria, in accordance with the result of family level taxonomic analysis. Besides them, five OUTs were classified into SAR92, and most of them were abundant in Group I and III. SAR92 has been shown to increase or become active during phytoplankton blooms (Teeling et al., 2012;


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Fig. 6. Relative abundance of the top 20 OTUs at each bacterial communities grouped by the T-RFLP profiles.

Wemheuer et al., 2014). We could think that these bacteria were also developed under influences of the phytoplankton bloom. On the other hand, SAR11 also appeared abundantly in Group I. However, SAR11 is well known to be a representative oceanic bacterial taxa distributing widely and abundantly in seawater of all over the world (Morris et al., 2002). Though SAR11 is often detected from estuarine water also, however, it has been shown that SAR11 is not an estuarine specific species (Fortunato et al., 2013) and that its activity (ratio of rRNA to DNA derived sequences) was decreased in estuarine waters (Campbell and Kirchman, 2013). Moreover, SAR11 was not detected as a major bacterial species from the neritic seawater collected at the Seto Inland Sea where influence of oceanic seawater was small (Taniguchi and Hamasaki, 2008). In this study, the relative abundance of SAR11 was the highest at Group IV which was distributed where the oceanic seawater intrusion was observed, suggesting that the SAR11 was derived from outside of the bay and that its abundance might have decreased while staying within the bay. Group III may be a mixture comprised of both surface (Group I) and bottom (Group II and IV) bacterial communities because hydrographic profiles indicate that stratification collapsed and vertical water mixing occurred at St. 7 where Group III was observed. Complementarily, the community diversity index and OTU compositions of Group III were similar to those of Group I. However, the ammonia concentration was high in the bottom water at St. 7 and the abundance of Shewanellaceae and Vibrionaceae tended to be high, although not statistically significant, in Group III. Effects of an inorganic nutrient supply caused by vertical water mixing may be different from those of phytoplankton bloom on bacterial community composition.

4.2. Effects from phytoplankton bloom sedimentation — Group II A unique bacterial community Group II was found in the bottom water of the coastal side of the bay where Chl. a concentration was high, probably because of the sedimentation of the phytoplankton bloom. This bacterial community was very diverse comparing to the other three communities. Delta and epsilonproteobacteria were observed considerably in the bacterial community. As sulfate-reducing bacteria are generally belongs to these subclasses, it seems that anaerobic micro-environment was formed on the degrading phytoplankton cells (Ploug et al., 1997), although the dissolved oxygen concentration was more than 70% of the saturation in the seawater. At a family level, Saprospira and Alteromonas appeared abundantly in Group II. Tada et al. (2011) has shown that Bacteroides, in which Saprospira was included, and Alteromonas had high activities in a phytoplankton bloom and that their activities were correlated to the organic matter concentration in the water, suggesting that these bacteria were increased quickly responding to the increase of organic matters. Bacteroides is also known to have particle attachment properties and abilities to degrade high molecular weight organic matters (Cottrell and Kirchman, 2000). Moreover, it has been shown that Saprospira was often isolated from epiphytic bacterial biofilms (Burke et al., 2011), and it had an algicidal activity on diatoms (Furusawa et al., 2003), and/or a protein degrading activity (Xia et al., 2014). These characteristics suggest that Saprospira/ Bacteroides ought to degrade the sinking phytoplankton cells in the bottom water at the expiry of phytoplankton bloom. Iamiaceae and Acidimicrobidae_incertae_sed which are classified to Actinobacteria were also abundant in Group II. Actinobacteria are generally found



in terrestrial soil and fresh water but are less abundant in marine environments (Fuhrman and Hagström, 2008; Campbell and Kirchman, 2013). A large amount of the water from melted snow is discharged into the Sendai Bay in the spring through the Abukuma river and others. Although, judging from the salinity profile, a massive influence of fresh water was not apparent at the bottom layer during sampling, some bacterial species of terrestrial origin may occur in Group II. Alternatively, the Actinobacteria might be marine species that have evolved to degrade sinking phytoplankton cells, because known cultivable relatives of Actinobacteria have been reported to decompose diverse large molecules (Goodfellow and Haynes, 1983). Bacterial community is often more diverse in bottom sediments than in seawater (Gobet et al., 2012; Aravindraja and Viszwapriya, 2013). Because a portion of bacteria in sediments should be raised up into overlying water by a bottom current, some bacterial taxa found specifically in Group II might be derived from the underlying sediment. The diverse bacterial assemblage should degrade phytoplankton cells effectively in the bottom waters. In addition, Flavobacteria are known to be the bacterial taxa which occurred characteristically during phytoplankton blooms (Pinhassi et al., 2004). However, the abundance of Flavobacteria was less in Group II than in Group I. This result suggests that a bacterial community which is developed in phytoplankton blooms may be different from that sedimentated to a bottom layer. 4.3. Effects from the outside of the bay — Group IV A distinctive bacterial community Group IV was observed in the deep water near the border with the outside of the bay where an intrusion of oceanic seawater was observed, implying that the bacterial community was derived from outside of the bay. This idea is also supported by the result that relative abundance of SAR11 was the highest in Group IV, because SAR11 is representative oceanic bacterial taxa. Besides SAR11, Burkholderiaceae and Rhodospirilaceae appeared characteristically in Group IV. Though Burkholderiaceae has been shown to be one of the indicator bacterial taxa of river and estuarine water (Campbell and Kirchman, 2013), Burkholderiaceae-related phylotypes also found in methane-rich marine environments such as deep subsurface sediments (Lanoil et al., 2001). A metagenomic analysis of methane vent microbial community in deep seawater indicated that Burkholderiaceae might get energy by denitrification (Pernthaler et al., 2008). One of the major OUTs (OTU16) which was classified into Burkholderiaceae had a high similarity (100%) with Ralstonia sp.; a kind of Ralstonia (Ralstonia eutropha) is known to have a gene relating to denitrification process. The nitrate concentration was high (8.4–11 μM) in the water where the OUT16 appeared abundantly, implying a potential of denitrification by the bacterial lineage. An unclassified gammaproteobacterial OUT (OTU15) also appeared abundantly in Group IV. Similar sequences were detected from coastal water of Arctic and subarctic north Pacific areas (Grzymski et al., 2012; Allers et al., 2013), suggesting that it might be a cold water species. 5. Conclusions Three distinctive bacterial communities were observed at the expiry of spring bloom in the Sendai Bay. The dominant one was distributed in the surface and middle layers of the bay, where inorganic nutrients were exhausted and bacterial abundance was high. Rhodobacteriaceae and Flavobacteria were the characteristic bacterial taxa in this bacterial community. The second one was distributed at the bottom layer at the coastal side of the bay where high Chl. a concentration was observed. This bacterial community was diverse and comprised of such bacterial taxa as Alteromonas and Saprospira which were thought to be related with phytoplankton cell degradation. The third one was distributed at the deep water near the border with the outside of the bay where oceanic seawater intrusion was observed. The relative abundance of SAR11 was the highest in this bacterial community, suggesting that this

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