Bacterial community structure along the subtidal sandy sediment belt of a high Arctic fjord (Kongsfjorden, Svalbard Islands)

Science of the Total Environment 619–620 (2018) 203–211

Contents lists available at ScienceDirect

Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Bacterial community structure along the subtidal sandy sediment belt of a high Arctic fjord (Kongsfjorden, Svalbard Islands) Antonella Conte a, Maria Papale a, Stefano Amalfitano b, Anu Mikkonen c, Carmen Rizzo a, Emilio De Domenico a, Luigi Michaud a, Angelina Lo Giudice a,d,⁎ a

Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy Water Research Institute, National Research Council (IRSA-CNR), Monterotondo, Rome, Italy c Department of Biological and Environmental Sciences, University of Jyvaskyla, Jyvaskyla, Finland d Institute for the Coastal Marine Environment, National Research Council (IAMC-CNR), Messina, Italy b

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Subtidal sands tackle, filter and regulate the transport of glacial inputs in fjords. • Sand deposits may house abundant and metabolically active microbial communities. • The bacterial community structure changed markedly along a glaciomarine gradient. • Melting ice and terrestrial runoff were main factors driving bacterial diversity. • Coastal sands can play a role in biogeochemistry and ecology of cold environments.

a r t i c l e

i n f o

Article history: Received 26 July 2017 Received in revised form 7 November 2017 Accepted 7 November 2017 Available online xxxx Editor: D. Barcelo Keywords: Coastal sands Glacial inputs Prokaryotic abundance Microbial community composition Ion PGM sequencing

a b s t r a c t Open fjords are subject to contrasting environmental conditions, owing to meltwater glacial inputs, terrestrial runoff, and marine water mass exchanges, which are exacerbated by anthropogenic and climate perturbations. Following a slope-dependent water circulation, the subtidal sandy sediment belt regulates the convergent transport of nutrients downward the fjord depths, and the effective entrapment of suspended particles and microorganisms. In this study, we aimed at testing how glacial and seawater inputs may influence the bacterial community structure of subtidal sand deposits in the Kongsfjorden. Through total and viable cell counting and an amplicon sequencing approach, we found relevant differences in bacterial community structure along the glacio-marine sampling transect. Viable and high nucleic acid content (HNA) cells represented an important fraction of the total community, generally decreasing toward the glacier front. Besides the predominance of Alphaand Gammaproteobacteria, Bacteroidetes, Firmicutes and Parcubacteria, the bacterial community structure was likely affected by the glacial activity in the inner fjord, with the occurrence of distinctive phylotypes belonging to Gemmatimonadates, Nitrospirae, Acidobacteria, and Chloroflexi. Overall, our outcomes highlighted that exploring the bacterial community distribution and structure can provide new insights into the active role of sand deposits in coastal cold environments. © 2017 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Institute for the Coastal Marine Environment, National Research Council (IAMC-CNR), Spianata San Raineri 86, 98122 Messina, Italy. E-mail address: [email protected] (A. Lo Giudice).

https://doi.org/10.1016/j.scitotenv.2017.11.077 0048-9697/© 2017 Elsevier B.V. All rights reserved.

204

A. Conte et al. / Science of the Total Environment 619–620 (2018) 203–211

1. Introduction With their extraordinary hydrological and biogeochemical properties, open fjords are very suitable natural laboratories for comparative studies on meltwater-dominated glaciomarine processes, which occur in Northern to Southern Polar Regions with contrasting levels of human disturbances (Dalpadado et al., 2016; Cottier et al., 2005). Steep environmental gradients and water mass exchanges across shelf–fjord boundaries have direct consequences on local geomorphological and ecological dynamics. Thus, environmental perturbations resulting from global climate change are likely to strongly affect the fjord ecosystem processes, with increasing meltwater outflow and intensive input of nutrients and fine-grained mineral material to the coastal marine environment (Svendsen et al., 2002; Willis et al., 2006). Mostly during summer, glacial meltwaters and terrestrial runoff from melting ice and snow were reported to stimulate biological activities in fjord waters (e.g., phytoplankton blooms), and to accelerate the accumulation rates of detritus and organic carbon burial in deep deposits (Piquet et al., 2010; Keil, 2015; Bourgeois et al., 2016). By converging downward to the fjord depths, the meltwater flow promotes geomorphic erosion activity and sediment transport, then determining local deepening of the glacier bed and steepening of the downglacier sides (Alley et al., 2003). Thus, following a slopedependent mass-transport model, the mean grain size of fjord sediments decreases with distance from the glacier heads. As documented for major fjord systems worldwide (Lindhors and Schutter, 2014), the sandy sediment belt may act as a filter system and regulate the convergent transport of inorganic/organic compounds (i.e., including nutrients and oxygen), and the effective entrapment of suspended particles and microorganisms (Hugh and Peter, 2013). Although larger grain size results in less surface area and lower organic carbon concentrations (i.e., per dry weight unit) available for epipsammic (i.e., attached to or moving through sand particles) microbial growth than those of fjord deep deposits, sandy sediments may house metabolically active microbial communities, with organic matter remineralization and oxygen uptake rates comparable to those estimated in organic-rich muddy sediments (Hargrave et al., 2008). The structure and function of marine sediment microbiomes are generally considered sensitive to changes in water physical-chemical properties (e.g., temperature, nutrient content, oxygen saturation, hydrodynamism, light patterns) and burial history on long-term timescales, which contribute in selecting the dominant bacterial species (Bouvier and del Giorgio, 2002; Sapp et al., 2007; Herlemann et al., 2011; Bjørlykke, 2014). Phylogenetic structure and metabolic properties of sediment bacteria were found to influence marine benthic processes in different ways, but a limited number of studies focused on sand deposits, although they cover up to 70% of coastal areas (Rusch et al., 2003; Ishii et al., 2004; Buhring et al., 2005; Musat et al., 2006). Particularly within fjord systems, the subtidal sandy sediment belt has thus far not been considered as a biologically active ecotone, although recent findings emphasized the importance of benthic primary production in Ccycling along the shoreline (Woelfel et al., 2010). Thus, a better understanding of the in situ bacterial community distribution and composition would gain new insights into the key role of sand deposits in such cold environments. The aim of this study was to explore the diversity patterns of sediment bacterial communities along the glacio-marine transition in a sub-Arctic fjord of the Svalbard archipelago. In particular, we entailed to investigate major shifts in the bacterial community structure of coastal sand deposits owing to the influence of prevailing glacial (inner fjord) or seawater inputs (outer fjord). 2. Materials and methods 2.1. Study site and sample collection The Kongsfjorden (79° N, 12° E) is an open glacial fjord located on the west coast of the Svalbard Archipelago. It is about 20 km long and

its width varies from 4 to 10 km (Svendsen et al., 2002). With an estimated volume of 29.4 km3, the internal water circulation is governed by an arm of saltier and warmer seawater, derived from the Atlantic Ocean and entering from the south. During summer, the water mass is mixed with cold and less salty water from melting glaciers. The outer fjord is influenced by oceanographic conditions and the inner fjord is influenced by large tidal glaciers (e.g. decreased salinity, increased turbidity, and decreased light penetration) (Svendsen et al., 2002; Zhang et al., 2015). The Atlantic influence makes this fjord sub-Arctic rather than Arctic, as might be expected at this high latitude. Five sampling sites (named A, B, C, D and E) were selected to be representative of the glacio-marine transition along the southern coast of the Kongsfjorden, also in accordance to the consolidated knowledge on water circulation along the south coast of the fjord (Svendsen et al., 2002; Cottier et al., 2005; Willis et al., 2006) (Fig. 1). Sites A and E could be reached by an inflatable boat sailing from NY- Ålesund, whereas Sites B, C, D were only reachable by walking 1–2 h on the muddy shore, as scheduled within the limited daily time available on site. Samples of sandy sediments were collected manually in July 2009 from 50 to 60 cm depth, by using pre-sterilized polycarbonate buckets. Subtidal coastal sands were composed of moderately-sorted, matrix-supported gravels with a clast content of 30–50%. Water temperature ranged from 4 to 7.3 °C, O2 was in the range 9.9–12.5 ppm, while salinity was between 18.1 and 35.9 psu. pH was 8.4 at all sites (Supplementary Table S1). All samples were preliminary processed within approximately 2 h after sampling in the laboratory of the UK Arctic Research Station in Ny-Ålesund. 2.2. Microbial abundance estimation 2.2.1. Cell detachment from sediment particles The abundance of prokaryotic cells associated with sandy sediments was estimated by following procedures for cell detachment and counting described elsewhere (Amalfitano and Fazi, 2008). Briefly, 1 g of wet sediment was suspended in 1 × Phosphate Buffered Saline (PBS) amended with 0.1% (w/v; Sigma-Aldrich) sodium pyrophosphate, by 30 s of sonication at 160 W (Bandelin SonoPuls HD 200; Probe MS 72/ D), followed by 30 s of vortexing at medium speed and 30 s in ice (10 cycles). Aliquots of supernatant were diluted (1:10) in filtersterilized seawater, fixed with filter-sterilized formaldehyde solution (final concentration 2%), and kept at +4 °C until analyses. 2.2.2. Cell counting by flow cytometry Detached prokaryotic cells were quantified and characterized by the Flow Cytometer A50-micro (Apogee Flow System, Hertfordshire, England) equipped with a solid state laser set at 20 mV and tuned to an excitation wave length of 488 nm. The volumetric absolute counting was carried out by staining with SYBR Green I (1:10,000 final concentration; Molecular Probes, Invitrogen). A fixed threshold value of 10 fluorescence units (FU) was set on the green channel to exclude most of the background noise. The light scattering signals (forward and side light scatter), green fluorescence (535/35 nm), orange fluorescence (590/ 35 nm) and red fluorescence (N610 nm) were acquired for detecting the cytometric properties of single cells in the sediment suspensions. Samples were run at the lowest instrumental flow rate (1.4 μL min−1) in order to keep the number of total events below 1000 events s−1. An exclusion gate was applied to avoid visualization of abiotic particles characterized by low green and high red fluorescence. The acquisition time was set at 120 s and minimum 10,000 total detected events. On average, N 2000 events were counted in the gate specifically designed for detecting bacteria. Data were retrieved using the Apogee Histogram Software (v2.09). Beside the total cell counts (cells mL−1), we recorded the mean signal intensity of the forward scatter (proportionally related to cell size; absolute units – AU), the mean green fluorescence (related to per-cell content of nucleic acids; absolute fluorescence units – FU), and the percentages of cells with low and high nucleic acid content

A. Conte et al. / Science of the Total Environment 619–620 (2018) 203–211

205

Fig. 1. Sampling sites along the coastal subtidal sandy sediment belt of Kongsfjorden.

(LNA and HNA, respectively). As described in Amalfitano and Fazi (2008), LNA cells are generally retrieved within 10–100 green FU, while HNA cells show values higher than 100 green FU over the fixed threshold. 2.2.3. Viable counts For the enumeration of cultivable heterotrophic bacteria, serial dilutions of detached samples were prepared (1:10 and 1:100, using filtersterilized seawater) and 100 μL of each dilution was spread plated in triplicate on Marine Agar 2216 (MA, Difco). Plates were incubated in the dark at 4 °C for 1 month. Data obtained are expressed as CFU g−1 of wet sediment. 2.2.4. 16S rRNA gene amplicon sequencing and post-run analysis Sediment sub-samples were directly frozen at −20 °C. Once in lab, DNA was extracted in duplicate from 1 g of each sediment sample by employing the PowerSoil DNA extraction kit (MoBio Laboratories, Carlsbad, CA, USA) according to the manufacturer's instructions. DNA concentrations and purity were quantified by NanoDrop ND-1000 UV–vis Spectrophotometer (NanoDrop Technologies, USA). The V1-V2 region of bacterial 16S rRNA genes was amplified by PCR. In order to reduce potential barcode bias, a two-step PCR protocol was applied, consisting of a first step with conventional PCR primers and then using product of the first reaction as a template for the second PCR to add barcoded primers for Ion Torrent sequencing. Duplicate PCR reactions of 40 μL were set up at 0 °C under a PCR cabin by using 1 μL of extracted DNA, 0.4 μL of Phusion High-Fidelity DNA polymerase (2 U μL−1), 8 μL of Phusion buffer (5×), 1 μL of each dNTP (10 mM), 1 μL of SYBR Green I 1:10,000, 1 μL of each primer (10 μM). The universal

primers 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 338r (5′-GCT GCC TCC CGT AGG AGT -3′) were used. The amplification was performed according to the program: 1) 98 °C for 1 min; 2) 30 cycles at 98 °C for 10 s, 53 °C for 30 s and 72 °C for 60 s; 3) 72 °C for 10 min. Amplified products were visualized by agarose gel electrophoresis (1.5%, w/v), using 1 × SYBR Safe DNA gel stain (Invitrogen). The two reactions were pooled and set up under the same conditions to add Ion Xpress barcodes for sample read identification, and IonA and P1 sequences needed in template preparation. To 0.5 μL of pre-amplified DNA the components of the PCR mixture were added to a final volume of 20 μL: 0.2 μL of Phusion High-Fidelity DNA polymerase (2 U μL−1), 4 μL of Phusion buffer, 0.5 μL dNTPs (10 mM), 0.5 μL of SYBR Green I 1:10,000, 0.5 μL of each barcoded primer (10 μM). The reaction was carried out according to the program described above but with only 6 cycles. Amplified products were visualized by gel electrophoresis as described above. PCR products were purified using the Agencourt AMPure XP (Beckman Coulter, Inc.) system, according to the manufacturer's instructions, and then quantified using the Qubit dsDNA HS Assay Kit with Qubit Fluorometer 2.0 (Invitrogen, Thermo Fisher Scientific). 20 ng of each purified product was pooled for emulsion PCR with Ion PGM Template OT2 400 Kit. Sequencing was performed on an Ion Torrent Personal Genome Machine™ using the Ion PGM Sequencing 400 Kit and the Ion 314™ chip (all Ion Torrent reagents by Thermo Fischer Scientific) following manufacturer's protocols. The raw data were analyzed using the bioinformatics analysis software MOTHUR (version 1.39.5). Barcodes and primers were identified with maximum one base error and trimmed off. Total number of reads retrieved per sample ranged between 5542 (site D) and 8782 (site C). Reads were cleaned by length (reads shorter than 200 bp were

206

A. Conte et al. / Science of the Total Environment 619–620 (2018) 203–211

discarded) and by average quality ≥25 in a 10 nucleotide window. Following the trimming step, the number of good quality reads ranged between 1625 (site E) and 2855 (site C). Remaining sequences were aligned with the Silva v.123 reference files database. Reads were denoised using the pre.cluster command (Schloss et al., 2011) to remove sequences that were likely due to pyrosequencing errors and assemble reads which differed only by 2 nucleotides. Chimeric sequences were identified and removed (Edgar et al., 2011). Finally the sequences were classified against the Silva v. 123 database (Quast et al., 2013) and the distance matrixwas created to generate an OTU table (0.03) for the subsequent analyses. After rarefying the data, we calculated the Bray-Curtis distance matrix and the rarefaction curves for the five samples for a 0.03 distance cutoff (Schloss et al., 2009). Obtained OTUs were used to generate a Venn diagram that was performed by R software version 3.0.1, using the VennDiagram package (Chen and Boutros, 2011). 2.2.5. Statistical analyses The Principal Component Analysis (PCA), based on the correlation matrix, was run to graphically synthesize the microbial community structure at each sampling site by considering the relative abundance of all bacterial phyla (% of total reads), and the occurrence of LNA and HNA cells (% of total cells). The first two principal components (PC1 and PC2) were chosen with the goal of finding the best summary of the microbial diversity data using a limited number of PCs. All variables were normalized using division by their standard deviations. Shannon diversity index (H′) for each sampling site was calculated based on the total number of good quality reads, after normalization to the lowest number of reads (i.e., 1625 at site E). 3. Results 3.1. Total and viable cell abundance in sandy sediments The total prokaryotic abundance was on average 6.6 ± 4.3 × 10cells g−1, with highest values assessed in sediments collected at site E. The ratio between LNA and HNA cells ranged between 11.4 (A) and 0.7 (C). The mean value of green fluorescence was highest at E (323.5 FU) and lowest at A (108.0 FU). The mean value of forward light scatter was highest at A (35.0 AU) and lowest at C (20.0 AU). On average, HNA cells were bigger in size and about 4 times more fluorescent than LNA cells. Viable counts of heterotrophic bacteria on MA plates were between 1.1 and 184.8 × 105 CFU g−1 (site C and E, respectively), with a mean value of 60.7 × 105 CFU g−1 (Table 1). (See Table 2.) 7

3.2. Phylogenetic composition of the bacterial community The Ion Torrent sequencing of the V1-V2 region of the bacterial 16S rRNA gene generated a total of 33,174 sequence reads for the five barcodes. After quality check and removal of chimeras, 11,173 highquality sequences were obtained from the five samples. H′ reached the highest value (5.937) in A and the lowest (2.301) in C. The diversity index and the observed richness (OTUs) showed a general symmetric pattern as it is shown in the rarefaction curve (Supplementary Fig. S1).

3.2.1. Bacterial phyla Overall, 1930 OTUs belonging to 23 different bacterial phyla were retrieved from sediment samples, with predominance of Proteobacteria (45.7% of total sequences), followed by Parcubacteria, Firmicutes, Bacteroidetes and Actinobacteria (15.8, 12.2, 9.6 and 5.7%, respectively). Minor groups (i.e. Nitrospirae, Saccaribacteria, Chloroflexi, Acidobacteria) occurred at percentages between 0.1 and 1.5%. A number of reads (4.7% of total sequences) did not show significant similarity with database sequences and were grouped as unclassified bacteria. A closer analysis of the dominant phylum, namely the Proteobacteria, revealed that the majority of the representative reads in this phylum fell within the classes Gamma- (24.0% of total reads), followed by the Alpha- (14.0%), and Deltaproteobacteria (4.1%). The Beta- (1.1%) and Epsilonproteobacteria (0.5%) were less represented. Bacteroidetes, Actinobacteria, Chlorobi, Firmicutes, Parcubacteria, Planctomycetes and Saccaribacteria occurred at all sites, even if often at a low percentage (b 2%). The occurrence of bacterial phylotypes is shown in Fig. 2. At site A, the Gammaproteobacteria were equally dominant with the Bacteroidetes (17.9% vs 20.4%), followed by Alphaproteobacteria and Actinobacteria (12.3 and 9.3%, respectively). Some specific phylogenetic groups were observed only in sediments from the inner site A, i.e. the Nitrospirae (5.2%), Gemmatimonadates (1.1%), Armatimonadates (0.1%), Cyanobacteria (0.7%), Dictyoglomi (0.1%) and Spirochaetes (0.1%). Conversely, Chlamydiae (0.1%) occurred only at site B. Gammaproteobacteria were particularly abundant at sites B and D (41.4 and 30.0%, respectively), together with Parcubacteria (33.3 and 17.0%, respectively) and Alphaproteobacteria (10.1 and 18.1%, respectively). Sediments form site C showed the predominance of Firmicutes (58.1%), while Alphaand Gammaproteobacteria represented about the 12.0% of the bacterial community. At site E, Parcubacteria were slightly more abundant than Gamma- and Alphaproteobacteria (25.0, 18.1 and 16.1%, respectively), followed by Deltaproteobacteria (10.2%). 3.2.2. Bacterial genera From 2.4% up to 28.8% of the total high quality bacterial sequences were not classified at genus level. Most retrieved genera occurred at percentages below 1% (data not shown). Table 3 shows the relative abundance of bacterial genera in the range 1–60% of total quality reads. The genera Illumatobacter (among Actinobacteria), Sphingorhabdus (among Alphaproteobacteria), Methylophaga and Granulosococcus (both among Gammaproteobacteria) occurred at all sites (Fig. 3). Also, the genera Sphingorhabdus (among Alphaproteobacteria), Porticoccus (among Gammaproteobacteria), Desulfopila (among Deltaproteobacteria) and Acetobacterium (among Firmicutes) were well represented. Actinobacteria (i.e. genera Cryobacterium, Illumatobacter and Propionibacterium), Bacteroidetes (i.e. genera Balneola, Flavobacterium, Gillisia and Marinibacter), Alphaproteobacteria (i.e. genera Algimonas and Oceanibulbus), Epsilonproteobacteria (i.e. genus Sulfurimonas) and Nitrospirae (i.e. genus Nitrospira) represented only 1–4% of the total bacterial sequences. The genera Flavobacterium (among Bacteroidetes) and Nitrospira (among Nitrospirae) were exclusively retrieved in sediments from site A, while representatives of the genus Acetobacterium (among Firmicutes) occurred only in site C (57.9%) where they were predominant.

Table 1 Abundance (mean ± standard deviation) and cytometric properties of the prokaryotic communities per gram of wet sediments along shoreline sediments of the Kongsfjorden. Cytometric analysis Site

Sample ID

Viable counts (CFU g−1 × 105)

Total counts (cells g−1 × 106)

LNA cells (%)

HNA cells (%)

Forward scatter (FU)

Green fluo (FU)

Nielsenfjellet Haavimbfjellet Gluudneset Brandalpynten Strypbekken

A B C D E

13.5 ± 7.8 1.2 ± 0.2 103.0 ± 209.4 1.1 ± 0.1 184.8 ± 15.0

46.8 ± 0.4 46.6 ± 0.1 69.4 ± 8.5 30.2 ± 1.3 138.3 ± 0.6

92.0 58.9 46.7 41.2 45.8

8.0 41.1 53.3 58.8 54.2

35.0 23.5 22.5 20.0 27.0

108.0 191.0 238.0 242.0 323.5

A. Conte et al. / Science of the Total Environment 619–620 (2018) 203–211

207

Table 2 Data from the Ion Torrent sequencing. Sample

DNA concentration in natural samples (ng μL−1)

DNA concentration after PCR reaction (ng μL−1)

Pool DNA concentration (ng)

Read number

OTU number

A B C D E

0.03 0.3 4.36 0.9 7.79

3.38 5.64 5.28 5.18 6.13

20 20 20 20 20

2792 1888 2013 2855 1625

810 145 171 377 427

Overall, most Gammaproteobacteria were grouped in the marine methylotrophic group 3 (MMG3) which is related to the genus Methylophaga (10.2% of total sequences), resulting predominant at site B (30.3%). Members in the genera Paraglaciecola, Granulosicoccus, Halioglobus and Porticoccus also occurred, with this latter that was more abundant in sites D and E (5.1 and 8.0%). Alphaproteobacteria were mainly represented by five genera, among which the most abundant was Sphingorhabdus (3.4% of total sequences), with highest abundances determined in D and C (8.3 and 4.7%, respectively). Other well represented alphaproteobacterial genera occurring in sediment samples were Algimonas and Oceanibulbus (0.3 and 0.6% of total sequences, respectively). The genus Desulfopila among the Deltaproteobacteria occurred at sites D (1.7%), C (1.0%) and E (9.4%). Among the

Epsilonproteobacteria, the genus Sulfurimonas represented the 1.5% of sequences from site E. Most represented genera among the Bacteroidetes occurred at percentages below 4% of total sequences, with Gillisia, Flavobacterium and Maribacter that were generally more abundant in site A (2.9%, 2.7% and 1.2%, respectively), and Balneola in site B (3.1%). The Actinobacteria were mainly represented by three genera, i.e. Illumatobacter (range 0.4–3.8% of total sequences), Propinibacterium (range 0.1–2.5%) and Cryobacterium (range 0.1–1.2%). 3.2.3. Statistical analyses The PCA biplot allowed synthesizing the distribution patterns of major bacterial taxa that differently typify the microbial community structure at each sampling site (Fig. 4). The first component, accounting

Table 3 Bacterial genera retrieved in sandy sediment samples along the Kongsfjorden. Values were as the percentage of reads related to each genera on the total number of quality reads. Genera occurring at percentages below 1% at all sites are not reported.

208

A. Conte et al. / Science of the Total Environment 619–620 (2018) 203–211

Fig. 2. Relative abundance (%) of main bacterial phylogenetic groups as determined by the Ion Torrent analysis in sandy sediments of the Kongsfjorden. The number of OTUs per sampling site is in brackets.

Fig. 3. Venn diagram showing unique and shared OTUs among the five sampling sites selected along the sandy sediment belt of Kongsfjorden. The number of OTUs per sampling site is in bracket.

A. Conte et al. / Science of the Total Environment 619–620 (2018) 203–211

209

Fig. 4. Principal component analysis (PCA) biplot, based on the correlation matrix, of the relative abundance of bacterial taxa and LNA, HNA cytometric groups retrieved from sediments at each sampling site. The dot size is proportionally related to the total cell abundance, as assessed by flow cytometry. The histogram plots show the contribution of each variable to the ordination (vector projection values) expressed as the Pearson correlation with the PCA axes. The significant correlations between variables and PC scores are discriminated by the dotted lines (p b 0.05).

for most of the total variance (54.1%), clearly separated sediment at the glacial front (A) from all others that were increasingly exposed to seawater inputs. Notably, 11 bacterial phyla were positively correlated with PC1 and they were almost exclusively retrieved in A. The second component (18.5% of the total variance) further discriminated sediments from sites with higher exposure to seawater. In particular, among the major taxa, Alphaproteobacteria and Deltaproteobacteria showed a higher relative abundance in D and E.

melting ice and seawater intrusion (i.e., from A to E). LNA and HNA groups are considered as constitutive traits of microbial communities in a variety of aquatic environments, with HNA cells being bigger in size and metabolically more active than LNA cells (Lebaron et al., 2002; Amalfitano et al., 2014). The occurrence of LNA and HNA groups in different proportions was reported to reflect the activity state of the microbial communities, but also a different phylogenetic community composition (Vila-Costa et al., 2012; Schiaffino et al., 2013).

4. Discussion

4.2. Bacterial community composition at the glacial front

4.1. Patterns of total and viable cells within the sediment microbial community

Benthic studies conducted in the Kongsfjorden have mainly focused on deep sediments collected by research vessels (Prasad et al., 2014; Srinivas et al., 2009; Canion et al., 2013). As highlighted by the PCA and reported in other studies (Piquet et al., 2010; Bourgeois et al., 2016), the bacterial community structure appeared to be mainly affected by the input of glacial meltwaters and terrestrial runoff waters from melting ice. The peculiar and more diverse composition observed in sediments from site A (H′ = 5.937, with almost 30% of total sequences unclassified) was related to the occurrence of distinct phylotypes (e.g., Armatimonadetes, Cyanobacteria, Dyctioglomi, Gemmatimonadetes, Spirochaetae and Nitrospirae) and may be strictly dependent on the glacial activity in the inner fjord. Sequences related to Chloroflexi, Acidobacteria and Nitrospirae were particularly abundant (or exclusively present) in site A. The former phylum is common in sediments and aquifers worldwide (Hug et al., 2013). According to Teske et al.

Values of total and viable cell abundance were comparable and within ranges reported for other marine coastal sediments (Ravenschlag et al., 1999; Bourgeois et al., 2016). In particular, sediments from sites B, C and D showed low viable cell counts, even if total cell counts resulted higher. This finding could be partly explained by the notable occurrence of marine methylotrophs (e.g., Methylophaga spp., as highlighted by the Ion Torrent analysis), which hardly grow on agar plates under aerobic conditions. As assessed by flow cytometry, the subgroup of cells with a high content of nucleic acids (i.e., HNA cells) represented an important fraction of the total microbial community at all sampling sites except for that closest to the glacier front (i.e., site A). The mean per-cell content of nucleic acids (green fluorescence signal) increased following the environmental gradient overimposed by

210

A. Conte et al. / Science of the Total Environment 619–620 (2018) 203–211

(2011), the Chloroflexi are likely to contribute to the diversified repertoire of polysaccharide hydrolases in the Svalbard sediments. The presence of Acidobacteria typically found in soil systems and nitriteoxidizing Nitrospirae may indicate a terrestrial input and an active nitrification process. Betaproteobacteria are typically of freshwater origin and were more abundant at the inner site A (4.2%). Members in the Cytophaga-Flavobacteria group of Bacteroidetes play an important role in the detritus food chain and carbon cycling in aquatic ecosystems (Cottrell and Kirchman, 2000). These chemo-organotrophic bacteria were reported to be proficient in the uptake and degradation of complex dissolved and particulate organic matter (Abell and Bowman, 2006; Grossart et al., 2005). The recovery of flavobacterial sequences (i.e. genera Flavobacterium, Maribacter and Gillisia) was also related to the presence of phytoplankton or fresh phytodetritus on the sediment surface (Teske et al., 2011; Roh et al., 2013). The highest percentage of Bacteroidetes occurred in sediments from the inner site A, in accordance with literature findings form ice sheets and waters with high content of dissolved organic matter (DOM) (Brakstad et al., 2008).

oxidizing Sulfurimonas are reported to play a relevant role within the global sulfur cycle (Campbell et al., 2006). 5. Conclusion In this study, all quantitative data collected to characterize the sediment microbial community along the coastal belt of the Kongsfjorden indicated that subtidal sands might host an abundant and highly diverse microbial community. The subtidal sandy sediment belt of fjord systems is subject to variable glacial, terrestrial and marine inputs that differently affect the sediment microbial community structure along the glaciomarine transition. Our findings on the abundance of total and viable bacterial cells, together with the phylogenetic identification of distinctive phylotypes of glacial or marine origin, suggest that coastal sandy sediments may have an undervalued active role in regulating carbon and nutrient cycling and the quality of inorganic/organic particulate compounds converging downward the fjord bed. Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2017.11.077.

4.3. Bacterial community shifts along the glacio-marine transition Notably, the Shannon diversity decreased along the glacio-marine transition in the Kongsfjorden (i.e., from site A to site D), with only a small increase at the outer site E. As reported for Arctic sediments (Ravenschlag et al., 1999; Li et al., 2006; Tian et al., 2009), the predominance of Proteobacteria, in co-occurrence with the Bacteroidetes, Firmicutes and Actinobacteria, suggests the presence of nutrients for heterotrophic bacterial growth along the fjord. Gammaproteobacteria and Bacteroidetes are reported as predominant components in superficial sediments (Arnosti, 2008; Teske et al., 2011). Both phyla were frequently identified in glacial environments such as cryoconite holes (Edwards et al., 2011; Cameron et al., 2012), glacier-feed streams (Wilhelm et al., 2013), fjords and polar waters influenced by glacial meltwaters (Zeng et al., 2009, 2013; Piquet et al., 2010, 2011). Alphaproteobacteria seemed to occur at low percentages in surface marine sediments and mostly related to the genus Sphingorhabdus (Arnosti, 2008; Teske et al., 2011) that were retrieved at all sites in this study. Among Gammaproteobacteria, sequences related to Methylophaga (i.e., the Marine_Methylotrofic_group_3, MMG3) were dominant over the total sequences retrieved in sediments from sites B and D (30.3 and 19.4%, respectively), whereas they were less represented elsewhere (range 0.3– 5.1%). The occurrence of this group is also probably due to the presence of organic carbon, released by glacier melting and diluted along the fjord. As bacteria recently found in a broad range of anoxic environments, Parcubacteria was the second most abundant bacterial phylum in our samples. Their occurrence (mainly in sites B, D and E) suggests that sediments in the Kongsfjorden could experience anoxic conditions. This finding was reinforced by the high relative abundance of Firmicutes (up to 58% in site C), which were almost exclusively represented by the genus Acetobacterium, acetogenic bacteria retrieved in anoxic freshwater and marine sediments (Sattley and Magigan, 2007). Further, acetogenesis appears to be a significant process in cold anoxic environments (Sattley and Magigan, 2007 and references therein). Differently from the inner site A, the site E was characterized by the higher abundance of the genera Desulfopila (within Deltaproteobacteria) and Porticoccus, this latter representing a distinct phyletic line of Gammaproteobacteria. The Deltaproteobacteria occurred in all samples, as generally reported for marine sediments (Teske et al., 2011). Sequences related to Desulfopila spp. were retrieved also in sites C and D. This genus was isolated for the first time by Suzuki et al. (2007) in estuarine sediments subject to both marine and freshwater inputs. Among Epsilonproteobacteria, the genus Sulfurimonas occurred at the outer sites D and E. Sulfurimonas members are frequently isolated from marine sediments (Han and Perner, 2015) and their most widely shared feature is chemolithoautotrophy (Cai et al., 2014). Members of sulfur-

Conflict of interest The authors declare they have any conflict of interest. Acknowledgements We wish to thank the colleagues Stefania Giannarelli and Sandro Francesconi (University of Pisa) for assistance with sample collection. This research was supported by grants from the project PolyArc “Occurrence of PCB-degrading bacteria in relation to PCB contamination in coastal marine environments in Ny-Ålesund (Svalbard, Spitsbergen)” which was financed by the European Centre for Arctic Environmental Research in Ny-Ålesund (ARCFAC V; FP6) [grant no. ARCFAC-026129-2008-70]. References Abell, G.C.J., Bowman, J.P., 2006. Ecological and biogeographic relationships of class Flavobacteria in the Southern Ocean. FEMS Microbiol. Ecol. 51, 265–277. Alley, R.B., Lawson, D.E., Larson, G.J., et al., 2003. Stabilizing feedbacks in glacier-bed erosion. Nature 424, 758–760. Amalfitano, S., Fazi, S., 2008. Recovery and quantification of bacterial cells associated with streambed sediments. J. Microbiol. Methods 75, 237–243. Amalfitano, S., Del Bon, A., Zoppini, A., et al., 2014. Groundwater geochemistry and microbial community structure in the aquifer transition from volcanic to alluvial areas. Water Res. 65, 384–394. Arnosti, C., 2008. Functional differences between Arctic seawater and sedimentary microbial communities: contrasts in microbial hydrolysis of complex substrates. FEMS Microbiol. Ecol. 66, 343–351. Bjørlykke, K., 2014. Relationships between depositional environments, burial history and rock properties. Some principal aspects of diagenetic process in sedimentary basins. Sediment. Geol. 301, 1–14. Bourgeois, S., Kerhervé, P., Calleja, M.L., et al., 2016. Glacier inputs influence organic matter composition and prokaryotic distribution in a high Arctic fjord (Kongsfjorden, Svalbard). J. Mar. Syst. 164, 112–127. Bouvier, T.C., del Giorgio, P.A., 2002. Compositional changes in free-living bacterial communities along a salinity gradient in two temperate estuaries. Limnol. Oceanogr. 47, 453–470. Brakstad, O.G., Nonstad, I., Faksness, L.-G., et al., 2008. Responses of microbial communities in Arctic sea ice after contamination by crude petroleum oil. Microb. Ecol. 55, 540–552. Buhring, S.I., Elvert, M., Witte, U., 2005. The microbial community structure of different permeable sandy sediments characterized by the investigation of bacterial fatty acids and fluorescence in situ hybridization. Environ. Microbiol. 7, 281–293. Cai, L., Shao, M.-F., Zhang, T., 2014. Non-contiguous finished genome sequence and description of Sulfurimonas hongkongensis sp. nov., a strictly anaerobic denitrifying, hydrogen- and sulfur-oxidizing chemolithoautotroph isolated from marine sediment. Stand. Genomic Sci. 9, 1302–1310. Cameron, K.A., Hodson, A.J., Osborn, A.M., 2012. Structure and diversity of bacterial, eukaryotic and archaeal communities in glacial cryoconite holes from the Arctic and the Antarctic. FEMS Microbiol. Ecol. 82, 254–267. Campbell, B.J., Engel, A.S., Porter, M.L., et al., 2006. The versatile epsilon-proteobacteria: key players in sulphidic habitats. Nat. Rev. Microbiol. 4, 458–468. Canion, A., Prakash, O., Green, S.J., et al., 2013. Isolation and physiological characterization of psychrophilic denitrifying bacteria from permanently cold Arctic fjord sediments (Svalbard, Norway). Environ. Microbiol. 15, 1606–1618.

A. Conte et al. / Science of the Total Environment 619–620 (2018) 203–211 Chen, H., Boutros, P.C., 2011. VennDiagram: a package for the generation of highlycustomizable Venn and Euler diagrams in R. BMC Bioinformatics 12, 35. Cottier, F., Tverberg, V., Inall, M., et al., 2005. Water mass modification in an Arctic fjord through cross-shelf exchange: the seasonal hydrography of Kongsfjorden, Svalbard. J. Geophys. Res. 110, C12005. Cottrell, M.T., Kirchman, D.L., 2000. Natural assemblages of marine proteobacteria and members of the Cytophaga-Flavobacter cluster consuming low- and high-molecularweight dissolved organic matter. Appl. Environ. Microbiol. 66, 1692–1697. Dalpadado, P., Hop, H., Rønning, J., et al., 2016. Distribution and abundance of euphausiids and pelagic amphipods in Kongsfjorden, Isfjorden and Rijpfjorden (Svalbard) and changes in their relative importance as key prey in a warming marine ecosystem. Polar Biol. 39, 1765–1784. Edgar, R.C., Haas, B.J., Clemente, J.C., et al., 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27, 2194–2200. Edwards, A., Anesio, A.M., Rassner, S.M., et al., 2011. Possible interactions between bacterial diversity, microbial activity and supraglacial hydrology of cryoconite holes in Svalbard. ISME J. 5, 150–160. Grossart, H.-P., Levold, F., Allgaier, M., et al., 2005. Marine diatom species harbour distinct bacterial communities. Environ. Microbiol. 7, 860–873. Han, Y., Perner, M., 2015. The globally widespread genus Sulfurimonas: versatile energy metabolisms and adaptations to redox clines. Front. Microbiol. https://doi.org/ 10.3389/fmicb.2015.00989. Hargrave, B.T., Holmer, M., Newcombe, C.P., 2008. Towards a classification of organic enrichment in marine sediments based on biogeochemical indicators. Mar. Pollut. Bull. 56, 810–824. Herlemann, D.P., Labrenz, M., Jürgens, K., et al., 2011. Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea. ISME J. 5, 1571–1579. Hug, L.A., Castelle, C.J., Wrighton, K.C., et al., 2013. Community genomic analyses constrain the distribution of metabolic traits across the Chloroflexi phylum and indicate roles in sediment carbon cycling. Microbiome https://doi.org/10.1186/2049-2618-1-22. Hugh, F., Peter, T., 2013. Shifts in composition of microbial communities of subtidal sandy sediments maximise retention of nutrients. FEMS Microbiol. Ecol. 83, 279–298. Ishii, K., Mußmann, M., MacGregor, B., et al., 2004. An improved fluorescence in situ hybridization protocol for the identification of bacteria and archaea in marine sediments. FEMS Microbiol. Ecol. 50, 203–212. Keil, R., 2015. Carbon cycle: hoard of fjord carbon. Nat. Geosci. 8, 426–427. Lebaron, P., Servais, P., Baudoux, A.C., et al., 2002. Variations of bacterial-specific activity with cell size and nucleic acid content assessed by flow cytometry. Aquat. Microb. Ecol. 28, 131–140. Li, H.R., Yu, Y., Zeng, Y.X., et al., 2006. Phylogenetic analysis of bacterial diversity in Pacific Arctic sediments. Wei Sheng Wu Xue Bao 46, 177–183. Lindhors, S., Schutter, I., 2014. Polar gravel beach-ridge systems: sedimentary architecture, genesis, and implications for climate reconstructions (South Shetland Islands/ Western Antarctic Peninsula). Geomorphology 221, 187–203. Musat, N., Werner, U., Knittel, K., et al., 2006. Microbial community structure of sandy intertidal sediments in the North Sea, Sylt-Rømø Basin, Wadden Sea. Syst. Appl. Microbiol. 29, 333–348. Piquet, A.M.-T., Scheepens, J.F., Bolhuis, H., et al., 2010. Variability of protistan and bacterial communities in two Arctic fjords (Spitsbergen). Polar Biol. 33, 1521–1536. Piquet, A.M.-T., Bolhuis, H., Meredith, M.P., et al., 2011. Shifts in coastal Antarctic marine microbial communities during and after meltwater-related surface stratification. FEMS Microbiol. Ecol. 76, 413–427. Prasad, S., Manasa, P., Buddhi, S., et al., 2014. Diversity and bioprospective potential (coldactive enzymes) of cultivable marine bacteria from the subarctic glacial Fjord, Kongsfjorden. Curr. Microbiol. 68, 233–238.

211

Quast, C., Pruesse, E., Yilmaz, P., et al., 2013. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41, D590–D596. Ravenschlag, K., Sahm, K., Pernthaler, J., et al., 1999. High bacterial diversity in permanently cold marine sediments. Appl. Environ. Microbiol. 65, 3982–3989. Roh, S.W., Lee, M., Lee, H.W., et al., 2013. Gillisia marina sp. nov., from seashore sand, and emended description of the genus Gillisia. Int. J. Syst. Evol. Microbiol. 63, 3640–3645. Rusch, A., Huettel, M., Reimers, C.E., et al., 2003. Activity and distribution of bacterial populations in middle Atlantic bight shelf sands. FEMS Microbiol. Ecol. 44, 89–100. Sapp, M., Wichels, A., Wiltshire, K.H., et al., 2007. Bacterial community dynamics during the winter–spring transition in the North Sea. FEMS Microbiol. Ecol. 59, 622–637. Sattley, W.M., Magigan, M.T., 2007. Cold-active acetogenic bacteria from surficial sediments of perennially ice-covered Lake Fryxell, Antarctica. FEMS Microbiol. Lett. 272, 48–54. Schiaffino, M.R., Gasol, J.M., Izaguirre, I., et al., 2013. Picoplankton abundance and cytometric group diversity along a trophic and latitudinal lake gradient. Aquat. Microb. Ecol. 68, 231–250. Schloss, P.D., Westcott, S.L., Ryabin, T., et al., 2009. Introducing Mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75, 7537–7541. Schloss, P.D., Gevers, D., Westcott, S.L., 2011. Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies. PLoS One 6, e27310. Srinivas, T.N.R., Nageswara, Rao S.S.S., Reddy, P., et al., 2009. Bacterial diversity and bioprospecting for cold-active lipases, amylases and proteases, from culturable bacteria of Kongsfjorden and Ny-Ålesund, Svalbard, Arctic. Curr. Microbiol. 59, 537–547. Suzuki, D., Ueki, A., Amaishi, A., et al., 2007. Desulfopila aestuarii gen. nov., sp. nov., a gramnegative, rod-like, sulfate-reducing bacterium isolated from an estuarine sediment in Japan. Int. J. Syst. Evol. Microbiol. 57, 520–526. Svendsen, H., Beszczynska-Mmller, A., Hagen, J.O., et al., 2002. The physical environment of Kongsfjorden-Krossfjorden, an Arctic fjord system in Svalbard. Polar Res. 21, 133–166. Teske, A., Durbin, A., Ziervogel, K., et al., 2011. Microbial community composition and function in permanently cold seawater and sediments from an Arctic fjord of Svalbard. Appl. Environ. Microbiol. 77, 2008–2018. Tian, F., Yong, Y., Bo, C., et al., 2009. Bacterial, archaeal and eukaryotic diversity in Arctic sediment as revealed by 16S rRNA and 18S rRNA gene clone libraries analysis. Polar Biol. 32, 93–103. Vila-Costa, M., Gasol, J.M., Sharma, S., et al., 2012. Community analysis of high- and lownucleic acid-containing bacteria in NW Mediterranean coastal waters using 16S rDNA pyrosequencing. Environ. Microbiol. 4, 1390–1402. Wilhelm, L., Singer, G.A., Fasching, C., et al., 2013. Microbial biodiversity in glacier-fed streams. ISME J. 7, 1651–1660. Willis, K., Cottier, F., Kwasniewski, S., et al., 2006. The influence of advection on zooplankton community composition in an Arctic fjord (Kongsfjorden, Svalbard). J. Mar. Syst. 61, 39–54. Woelfel, J., Schumann, R., Peine, F., et al., 2010. Microphytobenthos of Arctic Kongsfjorden (Svalbard, Norway): biomass and potential primary production along the shore line. Polar Biol. 33, 1239–1253. Zeng, Y., Zheng, T., Li, H., 2009. Community composition of the marine bacterioplankton in Kongsfjorden (Spitsbergen) as revealed by 16S rRNA gene analysis. Polar Biol. 32, 1447–1460. Zeng, Y.-X., Zhang, F., He, J.-F., et al., 2013. Bacterioplankton community structure in the Arctic waters as revealed by pyrosequencing of 16SrRNA genes. Antonie Van Leeuwenhoek 103, 1309–1319. Zhang, T., Fei, W.N., Qin, Z.Y., et al., 2015. Diversity and distribution of fungal communities in the marine sediments of Kongsfjorden, Svalbard (High Arctic). Sci. Rep. 5, 14524.