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Environmental Microbiology Reports (2015)

doi:10.1111/1758-2229.12273

Seawater mesocosm experiments in the Arctic uncover differential transfer of marine bacteria to aerosols

Camilla Fahlgren,1 Laura Gómez-Consarnau,1† Julia Zábori,2 Markus V. Lindh,1 Radovan Krejci,2 E. Monica Mårtensson,2,3 Douglas Nilsson2 and Jarone Pinhassi1* 1 Centre for Ecology and Evolution in Microbial model Systems – EEMiS, Linnaeus University, Barlastgatan 11, SE-39182 Kalmar, Sweden. 2 Department of Analytical Chemistry and Environmental Science and the Bert Bolin Centre for Climate Research, Stockholm University, Svante Arrhenius väg 8, SE-11418 Stockholm, Sweden. 3 Department of Earth Sciences, Uppsala University, Villavägen 16, SE-75236 Uppsala, Sweden. Summary Biogenic aerosols critically control atmospheric processes. However, although bacteria constitute major portions of living matter in seawater, bacterial aerosolization from oceanic surface layers remains poorly understood. We analysed bacterial diversity in seawater and experimentally generated aerosols from three Kongsfjorden sites, Svalbard. Construction of 16S rRNA gene clone libraries from paired seawater and aerosol samples resulted in 1294 sequences clustering into 149 bacterial and 34 phytoplankton operational taxonomic units (OTUs). Bacterial communities in aerosols differed greatly from corresponding seawater communities in three out of four experiments. Dominant populations of both seawater and aerosols were Flavobacteriia, Alphaproteobacteria and Gammaproteobacteria. Across the entire dataset, most OTUs from seawater could also be found in aerosols; in each experiment, however, several OTUs were either selectively enriched in aerosols or little aerosolized. Notably, a SAR11 clade OTU was consistently abundant in the seawater, but was recorded in significantly lower proportions in aerosols. A strikingly high proportion of

Received 21 February, 2014; revised 12 January, 2015; accepted 30 January, 2015. *For correspondence. E-mail [email protected]; Tel. (+46) 480 446212; Fax (+46) 480 447305. †Present address: Departament de Biologia Marina i Oceanografia, Institut de Ciències del Mar, CSIC, ES-08003 Barcelona, Catalunya, Spain.

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd

colony-forming bacteria were pigmented in aerosols compared with seawater, suggesting that selection during aerosolization contributes to explaining elevated proportions of pigmented bacteria frequently observed in atmospheric samples. Our findings imply that atmospheric processes could be considerably influenced by spatiotemporal variations in the aerosolization efficiency of different marine bacteria.

Introduction Sea spray from the surface ocean is one of the major contributors to natural aerosol mass fluxes on a global scale (Blanchard and Syzdek, 1970; 1982; O’Dowd and de Leeuw, 2007). During sea spray formation, wind forcing produces bubbles that burst and eject droplets or aerosols that influence atmospheric properties. Sea salt particles scatter solar radiation and increase albedo (Winter and Chýlek, 1997). In addition, marine aerosols may have a role as cloud condensation nuclei or as ice nuclei (Guriansherman and Lindow, 1993; Junge and Swanson, 2008; Orellana et al., 2011). The importance of the physical and chemical characteristics of abiotic aerosols has long been studied, but the corresponding influence of biological components has only recently attracted more focused attention (Lohmann and Leck, 2005; Despres et al., 2012). Moreover, increased attention is being given to how aerosolization can influence the distribution of biological aerosols in the atmosphere and how this contributes to the biogeography of microorganisms (Hervas et al., 2009; Fahlgren et al., 2010; Despres et al., 2012; Fröhlich-Nowoisky et al., 2012; Mayol et al., 2014). Due to their surface properties, biological aerosols are expected to be especially important for catalyzation of nucleation events at higher temperatures (above −15°C) than mineral or carbonaceous particles, but such impacts on global precipitation patterns remain largely unexplored (Phillips et al., 2008). Since the ocean surface layer is highly enriched in organic matter and microorganisms, aerosols from seawater can be efficient in transferring organic material to the atmosphere (Blanchard, 1989; Orellana et al., 2011). In the Arctic, it was recently shown that marine organic matter in the form of microgels can act

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C. Fahlgren et al. Aerosol collection point: gelatin & PTFE filter holder

A

Aerosols out OPC Clean air in; GF/C filter

Water in

Water jet stream Air

Pump

Seawater Water out

B

1

dN/dD cm-3 µm-1

0.1

0.01

as efficient cloud condensation nuclei (Orellana et al., 2011), while work on the marine model diatom Thalassiosira pseudonana showed that phytoplankton can act as efficient ice nucleation particles (Knopf et al., 2011). Several bacterial species are known that catalyse ice formation and induce precipitation, e.g. Xanthomonas spp., Pseudomonas syringae and Pseudomonas fluorescens (Maki et al., 1974; Maki and Willoughby, 1978; Kim et al., 1987), wherefore some of them are commercially used for lowering the energy demands during the production of artificial snow. Taken together, these reports indicate that a better characterization of the microbial content in the atmosphere is necessary for understanding the significance of bioaerosols in nucleation processes that contribute to regulate climate. The Arctic region is expected to undergo dramatic changes caused by global warming. With the predicted temperature increase (at a rate two to three times that of the global average temperature estimate), the region is estimated to be ice-free by summer of 2100 (Arzel et al., 2006). A consequence of this process could be an increased aerosol load to the atmosphere that would strongly affect cloud albedo (Struthers et al., 2011). How such changes will affect the potential transfer of bacteria to the atmosphere is unknown, and a characterization of the composition of present microbial communities is necessary. The aim of the present study was to determine if, and to what extent, different marine bacteria are selectively ejected from seawater when aerosols are formed during bubble bursting. Seawater samples were collected at three locations in Kongsfjorden, Svalbard [outer Fjord (Arctic Ocean), centre of the Fjord and close to the glacier front]. The bacterial community composition in experimentally generated aerosols and the source seawater was determined using both culture independent (16S rRNA gene clone libraries) and traditional culturing methodologies. Results and discussion

0.001

0.0001 20

100 1000 Bubble diameter (µm)

Fig. 1. Experimental set-up and bubble spectra for the experiments in Kongsfjorden, Arctic Ocean. Artificial sea spray experimental tank set-up (A). A water stream jet with recirculated seawater from the tank created bubbles and aerosols. Bubble size distribution in artificial sea spray experiment (B). Green line shows the power law of bubble spectra with a slope b = 2, characterizing a breaking wave (Medwin and Breitz, 1989). Blue line represents bubble spectra generated in this study. OPC, optical particle counter.

An important question regarding the formation of bioaerosols from the surface ocean is whether specific microorganisms are transferred to the atmosphere in a proportion similar to their proportion in the seawater source, or if there is some selection during the aerosol formation process. Therefore, we carried out a series of experiments using an impingement jet for the generation of sea spray aerosols. This method produces bubble spectra that are representative of those from natural oceanic waves (Fuentes et al., 2010; Hultin et al., 2010), and the data on bubble spectra in the experiments reported here were in agreement with these findings (Fig. 1B). The same experimental approach has produced

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports

Bacteria in Arctic marine aerosols important input data for modelling studies of the contribution of aerosols to the atmosphere and its effects on atmospheric cycles. Nevertheless, it has been used only occasionally to determine the transfer process of specific biological material to the atmosphere (Hultin et al., 2011). Our experimental approach addressed this question by experimentally investigating the transfer process of microorganisms from the ocean surface seawater. Clone libraries and community structure analysis Ten 16S rRNA gene clone libraries were constructed from samples collected at the three stations in the Fjord: six libraries from seawater samples and four from aerosol samples (Table S1). This resulted in a total of 1294 sequences. Grouping of clone sequences into operational taxonomic units (OTUs) based on a > 97% sequence identity level resulted in a total of 183 OTUs. Among these, 149 OTUs were of bacterial origin (1052 sequences), and 34 OTUs belonged to phytoplankton chloroplast/mitochondria or cyanobacteria (242 sequences). Overall, the distribution of sequences among Bacteroidetes, Alphaproteobacteria and Gammaproteobacteria and chloroplasts was relatively similar between seawater and aerosol samples (Table 1), although Bacteroidetes were more common in the aerosol fraction (37% versus 27%) and Alphaproteobacteria were more abundant in seawater (22% versus 11%). The clone library coverage calculated based on the bacterial sequences (excluding chloroplast sequences) varied between 60% and 97% (Table S1). The Shannon diversity index varied between 1.4 and 3.3 (Table S1). The two coupled air and seawater samples from 28 August exemplified a case of high and similar diversity indexes of 3.1 and 3.3, while the two seawater samples collected at station C on different dates differed markedly with diversity indices of 2.8 and 1.8 (Table S1). Dendrogram analysis of bacterial community composition showed that the aerosol and seawater communities clustered separately in three out of four experiments (Fig. 2). Interestingly, the dendrogram showed that the aerosol sample from station A on 28 August was positioned in the same subcluster as its corresponding seawater sample, along with the seawater sample from station B on 3 September. The similarity of the seawater and aerosol samples from 28 August can also be seen by comparing the clone library size-normalized abundances of individual bacterial and phytoplankton OTUs in Table 1. Statistical analysis of bacterial and phytoplankton OTU distributions confirmed that microbial community composition was significantly different in the aerosol compared with seawater samples in three experiments [i.e. station A 31 August, station B 1 September, and station B 3 September; permutational analysis of variance

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(PERMANOVA); F = 1.94, df = 1, 4, P = 0.0117]. We have no direct explanation to why the transfer efficiency of bacteria from seawater to aerosols differed between experiments, but we observed that the microbial community composition of the source seawater also varied considerably between experiments. This implies that the efficiency with which microbes are transferred from seawater to the atmosphere is dependent on both the presence/absence of particular microbes in the water and possibly also on their abundance and/or physiological state. Distribution of bacterial clone sequences between aerosols and seawater Detailed analysis of the diversity data yielded some intricate information regarding the potential selection of marine bacteria in the transfer from the seawater to the aerosols. First, focusing on the abundant OTUs, among the total of 149 detected bacterial OTUs, only 19 OTUs were represented by ≥ 10 clone sequences (Table 1). The most abundant OTU, the SAR11 clade OTU1 (Alphaproteobacteria), was found in all aerosol and seawater samples (accounting for nearly 17% of the clone sequences). However, this OTU was generally 6- to 40-fold more abundant in seawater compared with aerosols, with the exception of station A on 28 August where it was found in similar proportions in the aerosol and seawater samples. Across all experiments, OTU1 was significantly more abundant in the seawater samples compared with aerosols (Student’s t-test, P < 0.01). Cytophaga sp. OTU3 represented 25% of the clone library sequences in the seawater at station A on 31 August (only 1% in the corresponding aerosol sample), but at station B on 1 September it instead accounted for 28% of the clone library sequences in the aerosol sample (0 sequences in the seawater sample). Polaribacter sp. OTU6 (Bacteroidetes) was also detected in all aerosol and seawater samples (Table 1). While the normalized abundance of OTU6 was twofold to fourfold higher in the seawater fraction compared with the aerosols at station B (central Fjord), the abundances at station A (outer Fjord) were similar in the two fractions. The Bacteroidetes OTU38 and OTU13 were primarily found in aerosol samples, but were occasionally found in seawater samples as well. Considering the summed occurrence of these abundant OTUs in the paired samples from each of the four experiments (i.e. a total of 59 comparisons of 533 clone sequences in 19 OTUs; data in Table 1), 27 different OTUs (46%) were found in both the seawater and the paired aerosol sample, while 17 OTUs (29%) were unique to aerosols and 15 OTUs (25%) were unique to seawater samples. Recently, Cho and Hwang (2011)

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports

Uncult. SAR11 (99%) Uncult. Bacteroidetes (99%) Polaribacter irgensii (97%) Uncult. Verrucomicrobia (99%) Uncult. Bacteroidetes (99%) Uncult. Gammaproteobacteria (99%) Flavivirga jejuensis (95%) Uncult. Bacteroidetes (99%) Haliea sp. DSW4-37 (96%) Uncult. SAR11 (99%) Sulfitobacter japonica (97%) Uncult. Bacteroidetes (100%) Rhodobacteriaceae IMCC1933 (98%) Uncult. SAR11 (100%) Uncult. Actinobacteria (99%) Uncult. Gammaproteobacteria (100%) Uncult. Bacteroidetes (99%) Uncult. Bacteroidetes (99%) Pseudoalteromonas elyakovii (100%)

OTU

1 3 6 148 38 29 18 13 34 5 90 19 82 4 174 74 23 33 36 Euk Euk Euk Cyano Euk Cyano Euk Euk Cyano –

Alpha CFB CFB Verruco CFB Gamma CFB CFB Gamma Alpha Alpha CFB Alpha Alpha Actino Gamma CFB CFB Gamma

Taxon

3 3 0 0 1 1 0.7 0 0 148

9 0 8 8 3 0 5 0.7 2 1 3 2 3 0 0.7 0.7 0.7 1 0

Air

4 3 0 0 4 1 0.7 0 0 145

12 0 8 6 0 0.7 5 0.7 2 0 2 0.7 2 0 0.7 0 1 1 0

SW

28 August

6 9 0 0 2 2 2 2 0 101

2 1 5 6 18 1 1 1 2 0 0 0 0 0 0 1 0 4 0

Air

0 0.7 3 4 0 0 0 6 0.7 142

39 25 3 0 1 0 0 0.7 0 4 0 2 0 3 0 0 0 0 0

SW

31 August

5 1 0 1 3 1 4 0 0 102

1 28 4 0 1 1 0 22 3 0 0 0 0 0 0 3 0 0 0

Air

6 0 0 10 0 0 0 0 0 49

41 0 16 0 0 0 4 0 0 2 0 8 0 6 0 0 4 0 0

SW

1 September

18 2 0 0 2 0 0 0 0 91

3 0 3 1 4 2 0 0 9 0 3 0 0 0 9 0 0 2 1

Air

0.6 0.6 1 0 0 1 0 0 0 162

17 0 6 12 0 3 5 0 2 2 4 3 7 2 0.6 0 1 0 1

SW

3 September

Station B

Relative abundance of specific OTUs (%)

– – – – – – – – – –

– – – – – – – – – – – – – – – – – – –

Air

3 1 1 0.9 0 0.5 0 0 4 219

18 0.5 2 3 0.9 9 0.9 0 0.9 2 1 1 0.5 0.9 0.5 0.9 1 0 3

SW

1 September

– – – – – – – – – –

– – – – – – – – – – – – – – – – – – –

Air

1 3 10 7 2 2 3 0 0.7 135

27 11 4 0 0 0.7 0.7 0.7 0 5 0 1 – 1 0 3 0 0 0

SW

2 September

Station C

51 31 23 22 17 13 12 11 10 –

217 80 66 55 31 30 29 27 24 24 21 20 19 15 12 12 10 10 10

No. Seq

a. Closest relative in GenBank is given either as closest taxonomically described species (if sequence identity ≥ 93%) or as closest uncultured environmental clone defined at class or phylum level. All OTUs identified by taxonomically described species also had matches towards uncultured environmental clones with sequence identities > 98%. Abundant OTUs were defined as those represented by ≥ 10 clone sequences. Values are for experimentally generated aerosols (Air) and the corresponding seawater (SW). Numbers denote relative abundance of OTUs, i.e. the number of sequences of the OTUs normalized to the total number of sequences per sample; values below 1% are given with one decimal. The total number of clone sequences per sample, i.e. the sum of clone sequences for both abundant and rare OTUs, is given in the final row ‘No. Seq. per sample’ (see Table S1 for further details). Values in column ‘No. Seq’ indicate the total number of sequences (individual clones) that were obtained for each OTU in the study. Alpha, Alphaproteobacteria; CFB, Bacteroidetes; Actino, Actinobacteria; Gamma, Gammaproteobacteria; Verruco, Verrucomicrobia; Cyano, Cyanobacteria; Euk, Eukaryote.

Phytoplankton OTUs 112 Micromonas sp. (98%) 81 Chrysochromulina sp. (98%) 95 Emiliania sp. (97%) 72 Uncult Cyanobacteria (99%) 103 Env. Clone (98%) 94 Halomicronema sp. (89%) 122 Thalassiosira sp. (99%) 208 Thalassiosira sp. 93%) 134 Halomicronema sp. (90%) – No. Seq. per sample

Closest relative in GenBank (percent identity)a

Station A

Table 1. Identity and distribution of abundant bacterial and phytoplankton OTUs.

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© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports

Bacteria in Arctic marine aerosols Station A 28 August

Air

Station A 28 August

SW

Station B 3 September SW Air Station B 3 September Station C 1 September SW Air

Station A 31 August

Air

Station B 1 September

Station C 2 September SW SW

Station A 31 August

Station B 1 September SW 0

0.2

0.4

0.6

0.8

Bray–Curtis dissimilarity Fig. 2. Dendrogram comparing the microbial community composition in the aerosol and seawater samples from Kongsfjorden. Samples from aerosols (Air) and seawater (SW) at the three investigated stations and different dates are indicated. OTUs representing both bacteria (149 OTUs) and phytoplankton (34 OTUs) were included in the cluster analysis. The dendrogram was generated by applying Bray–Curtis dissimilarities to arcsine transformed relative abundance OTU data.

studied the microbial diversity in seawater (44 clone sequences in 21 OTUs) and among airborne bacteria (33 clone sequences in 21 OTUs) over the East Sea, Korea. Incidentally, and although their data set was small compared with the present one, their finding that 42% of the bacterial OTUs were shared between seawater and aerosols is remarkably similar to our estimate of shared OTUs. Our results suggest that, although in a majority of cases OTUs present in the seawater are also found in the aerosols, at any particular time, many OTUs are either selectively enriched in aerosols or only little aerosolized. Thus, if the experiments were considered individually, one would conclude that distinct bacterial taxa differ markedly in their ability to be transferred to the atmosphere. However, considering the diversity in the four experiments together, it is striking to note that none of the abundant OTUs were unique to the aerosol samples – i.e. all abundant OTUs were found in at least one seawater sample (Table 1); only one OTU was unique to seawater (SAR11 clade OTU4). Taken together, these results imply that a majority of abundant bacterial taxa have the potential to be transferred to marine aerosols. However, the transfer efficiency from seawater to aerosols of any particular taxon differs substantially over time and space. Focusing the analysis on the distribution of sequences in all 149 bacterial OTUs together, a slightly different

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picture was obtained on whether OTUs were dominated by clone sequences from aerosol or seawater samples. A total of 33% of the OTUs contained sequences from the two sample types. The remaining 29% and 38% of bacterial OTUs contained sequences only derived from aerosol or seawater samples respectively. Similarly, 36% of the total set of 34 phytoplankton OTUs contained sequences from the two sample types. However, almost 47% of phytoplankton OTUs contained sequences only derived from seawater, whereas a smaller portion (17%) contained sequences from aerosol samples only. Thus, with respect to the less abundant OTUs (i.e. those represented by < 10 clone sequences), a high proportion was unique to either seawater or aerosols. This emphasizes that both sample size and the diversity in particular samples can largely affect conclusions made about the ability of particular bacteria to enter the atmosphere from seawater, i.e. if diversity is very high, each OTU will be represented by only few sequences and most likely only in one sample type.

Colony-forming bacteria and their pigmentation The number of bacteria able to form colonies on solid culture media [colony-forming units (cfu)] ranged from 0.3 to 2.8 × 103 cfu ml−1 in the seawater and 0.2–2.0 × 103 cfu m−3 in the aerosol samples (Table 2). The abundance of cfu in the aerosols showed no systematic variations over time or between the sampling stations (Table 2). The cfu abundance in the aerosols (median of 1.1 × 103 cfu m−3) is in the range of values reported in the literature for natural atmospheric environments. For example, during a seasonal study of airborne bacteria conducted on the Swedish east coast by the Baltic Sea, the median

Table 2. Abundance of colony-forming bacteria [colony-forming units (cfu)] in aerosols and seawater in the Kongsfjorden experiments.

Colony-forming unit abundance

Station A 28 August 31 August 2 September Station B 31 August 1 September 3 September Station C 2 September 3 September 4 September

Proportion of pigmented bacteria (%)

Aerosol (m3)

SW (ml)

Aerosol

SW

9.9 × 102 20.0 × 102 11.0 × 102

12.0 × 102 13.0 × 102 28.0 × 102

88 72 85

89 62 9

2.1 × 102 9.6 × 102 4.1 × 102

7.1 × 102 9.9 × 102 9.6 × 102

81 93 94

59 20 19

11.0 × 102 2.4 × 102 13.0 × 102

11.0 × 102 2.4 × 102 27.0 × 102

93 90 67

31 5 3

Also shown is the proportion of pigmented colonies among the cfu.

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A

B

C

D

Fig. 3. Agar plates with colony-forming bacteria from the aerosolization experiments. Two coupled samples with bacteria from seawater (A) and aerosol (B) samples from experiments with water from station C on 2 September. Control (blank) sample before generation of aerosols in the experimental tank (C), and subsequent aerosol sample after 3 h aerosol generation (D) with water from station B on 3 September.

concentration was 0.36 × 103 cfu m−3 (Fahlgren et al., 2010) while Fuzzi and colleagues (1997) and Kellogg and Griffin (2006) reported up to 104 cfu m−3. In a bubbling experiment carried out in the Baltic Sea, similar to that done here, the cfu numbers ranged from some hundreds up to 6.0 × 103 cfu m−3 (Hultin et al., 2011). The cfu abundances recorded here are noteworthy from two perspectives: (i) they represent the lower limit of abundance of marine bacteria that can be aerosolized under the physical conditions given by the experimental set-up (lower limit since bacteria unable to form colonies on the growth media used are not included in the estimate) and (ii) they show that a considerable number of bacteria survive the physical forcing involved in aerosol formation. It is frequently observed that a large fraction of cultured bacteria in aerosol samples are pigmented (Griffin et al., 2006; Fahlgren et al., 2010; Cho and Hwang, 2011), and it has been suggested that pigmentation may contribute to improving bacterial survival in the atmosphere (primarily since it provides some protection from DNA damaging UV light) (Tong and Lighthart, 1997a,b). Strikingly, the proportion of pigmented bacteria (e.g. yellow or orange) were significantly higher in the aerosol compared with seawater samples (Student’s t-test, P < 0.001), and accounted for between 67% and 94% of the cfu in the aerosols and down to 3% in the seawater (Table 2, Fig. 3). Unusually high proportions of

pigmented bacteria were found among the seawater cfu at station A on 28 August, so that remarkably similar values were recorded for seawater (89% pigmented) and aerosols (88% pigmented) on this date (Table 2). Interestingly, this sampling was also where the highest similarity between aerosol and seawater samples was found in the total DNA community analysis (Fig. 2, Table 1). Our results show that there is a strong selection for pigmented bacteria already in the transfer from seawater to aerosols. The reason for this selection remains unknown. Remarkably, major portions of these pigmented bacteria belonged to the Flavobacteriia (Bacteroidetes). In contrast to members of the SAR11 clade discussed below, the Flavobacteriia have a life strategy where particle attachment is important (Kirchman, 2002; Fernandez-Gomez et al., 2013). If it is this life strategy or the pigmentation itself, by influencing surface properties, that causes the large selection towards aerosolization remains unknown. However, irrespectively of the specific mechanisms, our results highlight the predisposition of pigmented bacteria to be ejected into the atmosphere in the first place. To identify the colony forming bacteria that dominated our samples, the 16S rRNA gene sequences of 29 seawater isolates and 35 aerosol isolates were determined (Fig. S1). The majority of bacteria isolated from seawater belonged to the Gammaproteobacteria (25 isolates) and Alphaproteobacteria (25 isolates). The aerosol samples contained similar numbers of Alphaproteobacteria compared with seawater (14 versus 10) while the number of Gammaproteobacteria was slightly lower than in the seawater (9 versus 17). Isolates belonging to the Bacteroidetes (six isolates) and Firmicutes (three isolates) were only found in the aerosols. In a total of six bacterial clusters, isolates from aerosols were identical to bacterial isolates and/or clones from seawater (Fig. S1; clusters marked by brackets). The bacteria isolated from the bubbling experiments mainly belonged to the same major groups as the OTUs detected by 16S rRNA gene clone sequencing (i.e. Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes). Nevertheless, at the level of specific taxa (approximately species level), there was only minor overlap between bacteria detected by culturing compared with culture independent 16S rRNA gene clone library analysis. Still, from among the 64 isolated bacteria, representing around 38 different taxa (i.e. single sequences or clusters sharing identical 16S rRNA gene sequences), at least six different bacterial taxa represented by sequences found in aerosols were also found by cloning. The major taxa detected in our aerosol samples, for example represented by genera like Pseudomonas (Gammaproteobacteria), Sphingomonas (Alphaproteobacteria) and Bacillus (Firmicutes), are frequently

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Bacteria in Arctic marine aerosols reported from natural atmospheric samples (Fuzzi et al., 1997; Amato et al., 2007; Brodie et al., 2007; Fahlgren et al., 2010). Our findings suggest that bubble bursting could be an effective mechanism by which they enter the atmosphere. Phytoplankton clone sequences Our clone libraries contained two distinct groups of OTUs with high similarity to chloroplast and mitochondrial 16S rRNA gene sequences (Fig. S2). Some of these OTUs, like those affiliated with Ochromonas sp., were detected both as mitochondrial and chloroplast sequences. The two most abundant phytoplankton OTUs were Micromonas sp. OTU112 and Chryschromulina sp. OTU 81, which were present in nearly all aerosol and seawater samples (Table 1). For Micromonas sp. OTU112, there was a tendency towards higher relative abundance in aerosols compared with seawater samples (Student’s t-test, P < 0.1). The finding of Thalassiosira sp. among the abundant OTUs (also in aerosols) can also be noted. Altogether, chloroplast-containing material made up a fair part of the clone libraries, constituting 8% and 12% of seawater and aerosol clone sequences respectively. In a previous study in the Fjord, chloroplast sequences accounted for up to 28% of the seawater clone sequences (Zeng et al., 2009). Since the 16S rRNA genes in chloroplasts and mitochondria are highly conserved, they can only be used to resolve phylogeny at a low taxonomic level (Soltis et al., 1998). Nevertheless, a few interesting notes on some of the abundant phytoplankton taxa can be made. First, the most abundant phytoplankton in our samples, Micromonas sp., is a ubiquitous picoeukaryote taxon found in surface seawater; this genus is today divided into five clades, one of which consists of low-temperature species only present in Arctic regions (Lovejoy et al., 2007; Foulon et al., 2008). In the present study, Micromonas sp. was found at all three stations from the glacier zone to the mouth of the Fjord. Notably, this abundant phytoplankton was efficiently transferred from seawater to aerosols. In contrast, the globally abundant coccolithophore Emiliania sp. was found in seawater samples but not in the aerosols. Lastly, the clone sequences affiliated with the marine diatom T. pseudonana were detected both in seawater and in aerosols. Consistent with our results, T. pseudonana was recently shown to very efficiently act as ice nuclei under typical tropospheric conditions (Knopf et al., 2011). The authors thus suggested that marine biogenic particles can play an important role in atmospheric ice nucleation – at levels similar to desert dust and anthropogenic organic particles – and in turn affect cloud processes and associated radiative balance (Knopf et al., 2011).

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Concluding remarks Several bacteria found in the experimental aerosols represent taxa that are commonly observed in natural atmospheric environments, such as Acinetobacter sp., Polaromonas sp., Methylobacterium sp. and Psychrobacter sp. However, bacteria in the abundant and widespread SAR11 clade, which typically account for major portions of cell numbers in the open ocean (Vergin et al., 2013), have to our knowledge not been detected in natural aerosol samples before (Brodie et al., 2007; Fahlgren et al., 2010; 2011; Urbano et al., 2011). This most likely reflects that previous studies used culturedependent methodologies that would not detect SAR11 bacteria since they do not form colonies on solid media. SAR11 clade bacteria are recognized as the smallest free-living bacteria yet known (biovolume of 0.03– 0.05 μm3) and for not actively seeking or attaching to each other or to particles (Giovannoni et al., 2005). These characteristics potentially contributed to the low incidence of detection of SAR11 clade bacteria in the aerosols compared with seawater. Still, in one experiment, the SAR11 OTU1 were detected in similar proportions in both the aerosol and seawater samples, suggesting the potential of these bacteria for spreading through the atmospheric environment. Experimental assessment of SAR11’s ability for long-term survival in aerosols would be welcome to validate if transport following aerosolization is part of their life history strategy. We conclude that miniscule size and a single-cell life strategy is no fundamental hinderance per se to marine bacteria in forming part of marine aerosols. Collectively, our results show that bacterial aerosolization is a widespread but selective process potentially acting on surface layer marine bacteria. Selectivity during aerosolization is determined by a number of linked mechanisms. In the context of this study, it remains unknown if the observed selectivity results from differential adherence of bacteria to bubbles when bubbles move through the water column, or if it results from differences in volatilization in the very moment of aerosol formation during bubble bursting. Given that our experiment mimicked aerosol-generating forces in the natural environment, aerosolization processes may selectively enrich for specific marine bacterial populations entering the atmosphere. These findings highlight the importance of unravelling the mechanisms and efficiency of transfer to aerosols of biological material in general and of marine microbes in particular. Future studies will need to focus on the physicochemical or morphological characteristics that may influence the success of particular bacterial taxa at being transferred to aerosols and how they survive in the atmosphere (e.g. cell size and density, presence of pigments, composition

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of the cell membrane), as they represent a potential to succeed in ‘invading’ new environments. Also, work using high-throughput next-generation sequence analyses or refined culturing methods will be valuable to retrieve information on the diversity and physiology on the as yet uncultured majority of microbes in aerosols. Moreover, from a climate perspective, further characterizations of the quantity and quality of biological material in the atmosphere, in relation to the surrounding environment or ecosystem, will detail the role of aerosol microbes in determining atmospheric processes and climate.

Experimental procedures Description of sampling location and seawater collection Kongsfjorden, Svalbard, is located at the meeting point of the Atlantic and the Arctic Ocean. The salinity of the surface water changes from 35.4 practical salinity units (PSU) in the outer parts of the Fjord to 28.0 PSU close to the glacier (Svendsen et al., 2002). In the innermost part of the Fjord, glacier sediment and freshwater discharge result in high seawater turbidity and salinity stratification (Svendsen et al., 2002). Seawater for experiments was collected at three locations in year 2009: outer Fjord (station A: 79°0′ N, 11°26′ E), central Fjord (station B: 78°94′ N, 11°96′ E) and inner Fjord/ glacier (station C: 78°53′ N, 12°29′ E). Water was collected from approximately 2 m depth (as representing water column surface mixed layer, without intents to include water directly from the seawater/atmosphere microlayer), and transported to the laboratory where it was transferred to the experimental tank within one hour after collection.

Experimental set-up For each experiment with water from one of the stations, a cylindrical stainless steel tank of approximately 180 l (Fig. 1A) was filled with 60 l of seawater. At the base of the tank, an outlet enabled sampling of seawater. The top of the tank was sealed with a gasket and a stainless steel lid. Seawater from inside the tank was recirculated by a pump to maintain an impingement water jet stream from the top of the tank – this water jet created bubbles in the water and generated aerosols in the air phase of the tank. These aerosols were sampled for aerosol chemistry and microbiology. Air inflow through glass fiber filters (GF/C) generated a positive pressure in the tank and prevented unintended inflow of outside air that would potentially contaminate the experimental aerosols. The positive pressure was used to collect aerosol particles that were generated inside the tank during 3 h. Aerosols were collected on filters placed in filter holders placed on the air outlet in the lid of the tank (see details below: Biological samples). Airflow was maintained around 240 l h−1, and was measured at the beginning and end of each experiment. Between each experiment, the experimental tank was cleaned with 70% ethanol and extensively rinsed three times with milli-Q water. Before each experiment, the tank was rinsed with sample water.

Bubble spectra Subsurface bubble spectra were characterized using the established Netherlands Organisation for Applied Scientific Research (TNO) Physics and Electronics Laboratory optical bubble measuring system (Leifer et al., 2003). The bubble-measuring device was mounted vertically in the tank. The water surface was ∼ 32 cm above the tank bottom, and was controlled by a gooseneck on the outside of the tank; the sampled volume was ∼ 4 cm under the water level. The water jet was mounted at about 14 cm above the water level with a water flow through the jet at 0.33 l min−1. According to a parameterized bubble spectra equation, natural wind-generated bubble spectra were described by Hultin and coleagues (2010). With a maximum number concentration at a diameter of about 60–80 μm and a falloff towards larger sizes that approaches the power law dN/dr = ar-b (Medwin and Breitz, 1989), where r is the bubble radius, the bubble spectra in the tank resembled the bubble spectra in a typical breaking wave at sea (Fig. 1B).

Biological samples Aerosol generation experiments with seawater from the Fjord were carried out twice for each of the three stations (Table S1). From both the seawater and the aerosols, samples were collected for cultivation studies (number of cfu per volume water or air, and phylogenetic identification and pigmentation of bacterial colonies) and for cultivationindependent analysis of bacterial community composition through collection of microbial biomass for subsequent DNA extraction and 16S rRNA gene clone library construction. For cfu enumerations in aerosols, gelatin filters (47 mm diameter, 3 μm pore size, Sartorius) were used. For bacterial community analyses in aerosols, polytetrafluoroethylene filters (PTFE, 47 mm diameter, 0.45 μm pore size, Pall Corporation) were used. At the initiation of each experiment, an optical particle counter was run for 20 min to control that there were no bubble formation or particles inside the tank before the water jet stream was started. During this time, control samples (i.e. blanks) were collected on gelatin filters and PTFE filters. Each experiment ran for approximately 3 h and, once terminated, 1 l of seawater was filtered onto Isopore membrane filters (GTTP, 47 mm, 0.2 μm pore size, Millipore) for subsequent culture independent analysis of bacterioplankton community composition. Due to the high turbidity of the seawater at station C, only 0.5 l of water from this station could be filtered.

DNA extraction and 16S rRNA gene clone library construction DNA was extracted from entire PTFE filters (for aerosol samples) and GTTP filters (for seawater samples) using the previously described hexadecyltrimethylammonium bromide protocol (Fahlgren et al., 2010). DNA extraction was successful from all six seawater samples; station C aerosol samples could not be extracted – possibly due to the high glacier sediment load at this station – resulting in a total of

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports

Bacteria in Arctic marine aerosols four successfully extracted aerosol samples. Moreover, no DNA was detected in extractions from the aerosol control samples (blanks) from experimental tank air filtered through PTFE filters before aerosol generation was initiated (consistent with the nearly complete lack of bacteria detected on agar plates from control gelatin filter samples; e.g. Fig. 3C). The 16S rRNA gene was amplified using 5 pmol each of the universal bacterial primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′TACGGYTACCTTGTTACGACTT–3′) (Lane, 1991). The polymerase chain reaction (PCR) was carried out using Illustra PuReTaq Ready-To-Go PCR Beads (GE healthcare Life Sciences) according to the manufacturer’s instructions. The PCR cycling conditions were: initial denaturation at 95°C for 2 min followed by 30 cycles consisting of 95°C for 30 s, 50°C for 30 s, 72°C for 45 s and a final elongation at 72°C for 7 min. The size of the amplified products were verified on a 1% agarose gel and thereafter cloned using TOPO TA Cloning kit (Invitrogen). Stab cultures were created by transferring 96 clones into a 96-well plate containing Luria–Bertani medium complemented with 150 μg ml−1 ampicillin, which were sent to GATC Biotech, Germany (plate supplied by GATC) for sequencing. The clones were sequenced by Sanger sequencing using the 27F primer, which resulted in sequences with an average size of 514 bp and a median of 522 bp. Between 49 and 219 16S rRNA gene, clone sequences were obtained from each of the 10 successfully analysed samples (see Table 1 and Table S1 for details).

Colony-forming bacteria and 16S rRNA gene sequencing of bacterial isolates Aerosol samples and controls collected on gelatin filters were dissolved in 4 ml of 0.2 μm pore size filtered and autoclaved seawater. A total of 100 μl of the bacterial suspension was spread in triplicates on Marine Agar 2216 (Difco; the growth-promoting substrates in this seawater agar plate medium is principally peptone and yeast extract). The plates were incubated at room temperature for 14 days (to ensure detection of both fast and slow growing bacteria). Once growth was observed, the number of cfu was determined every third day, along with visual recordings of the bacterial colony morphology and pigmentation. For cfu in the seawater, 100 μl of water from the experimental tank was spread on marine agar plates in triplicates. For the identification of cfu, colonies were randomly isolated from the aerosol and seawater samples. Bacterial isolates were streaked on new agar plates for purification. Between 5 and 15 different bacteria were isolated from each experiment, with the higher number from experiments where the diversity of colony morphologies was higher. Genomic DNA from the isolates was extracted using the EZNA Tissue DNA Kit (Omega Bio-Tek). Amplification of the 16S rRNA gene by PCR was done as described above for the clone libraries. PCR products were run on an agarose gel to control the size of the amplified fragment. The PCR products were purified using EZNA Cycle-Pure Kit (Omega Bio-Tek) and sequenced using Sanger sequencing with the universal bacterial forward primer 27F at Macrogen Corporation (Seoul, Korea).

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Sequence analyses The 16S rRNA gene sequences reported here have been deposited in GenBank under the accession numbers JQ858523-JQ859816. The quality of retrieved sequences was controlled using the freeware programme 4Peaks 1.7.2 (developed by A. Griekspoor and T. Groothuis). Sequences were clustered into OTUs with > 97% sequence identity, using Seqman II (Lasergene v. 7, DNAstar). The taxonomic affiliation of individual OTUs was determined by Basic Local Alignment Search Tool (BLAST) searches (Altschul et al., 1990), using blastn with our 16S rRNA gene sequences as queries against the nr/nt nucleotide collection in GenBank (National Center for Biotechnology Information; December 2014 release). For further taxonomic exploration, also BLAST searches against the GenBank nr/nt nucleotide collection were done using the Entrez Query search term ‘bacteria[ORGN] AND sp nov[WORD]’ to specifically identify the closest taxonomically described bacterial species. Assignment to taxa of our OTUs was based on the percentage of sequence identity against the top 10 hits in each of these searches. Maximum likelihood phylogenetic trees were constructed using the freeware programme MEGA, version 4.0 (available at http://www .megasoftware.net) (Tamura et al., 2007). Good’s coverage index was used to determine the clone coverage based on bacterial sequences (Good, 1953). Shannon diversity index was calculated using H′ = −Σipilnpi, where pi is the proportion of an OTU in a sample (i.e. pi = ni/N, where n is the number of clone sequences of an OTU and N is the total number of clone sequences in the sample) (Shannon, 1948). The diversity index was calculated for a dataset where each sample was subsampled to n = 63 bacterial 16S rRNA gene clone sequences. The community composition of bacterial assemblages in the seawater and in the generated aerosols of experiments from the different Fjord stations was compared by cluster analysis, whereby a dendrogram (hierarchical similarity tree) was generated by applying Bray–Curtis dissimilarities and using the package Vegan (Oksanen et al., 2010) in R version 3.1.2. Prior to cluster analysis, relative abundance OTU data were arcsine transformed to reach normal distribution. The cluster analysis was based on the abundance of OTUs in the different samples, where relative OTU abundance was given by the number of clone sequences in each OTU in relation to the total number of clones in each sample. For construction of the dendrogram, chloroplast sequences were included in the analysis; exclusion of the chloroplast sequences resulted in the same branching pattern (data not shown).

Statistical analyses For differences in microbial community composition between aerosols and seawater, PERMANOVA was carried out on the four dates where linked aerosols and seawater samples were obtained (i.e. station A on 28 August and 31 August, and station B on 1 September and 3 September). Input data consisted of the clone library size-normalized abundance of all 149 bacterial OTUs and the 34 phytoplankton OTUs. Student’s two-tailed t-tests were done to evaluate potential differences in distribution between aerosols and seawater samples of the dominant bacteria and phytoplankton OTUs, using their clone library size-normalized abundance. A

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports

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C. Fahlgren et al.

Student’s two-tailed t-test was also done to determine potential differences in distribution between aerosols and seawater samples of pigmented compared with non-pigmented cfu. For the statistical analyses, P-values < 0.05 were considered as significant.

Acknowledgements Sabina Arnautovic is acknowledged for skillful assistance in sample processing. We thank Åke Hagström for valuable discussions, and José M. González for contributing stringent support on bioinformatics analysis. We thank Gerrit de Leeuw for providing the TNO optical measuring system, and reviewers on past and present versions of the manuscript for thoughtful comments and corrections. This experimental work was part of the interdisciplinary climate project GRACE (GReenhouse Arctic ocean and Climate Effects of aerosols) financed by the Swedish Research Council (VR; contract no 2007-8362), and a collaboration between four departments at Stockholm University and the Linnaeus University, with additional coworkers from Gothenburg University, as well as Finland, Norway, Germany and the Netherlands. This study was further supported by grants from the Swedish Research Council VR and the Swedish Research Council FORMAS programme EcoChange to JP, and the FORMAS research grant ‘Seasonal variation in the primary marine aerosol source due to physical and bio/chemical processes’ (Grant No. 2007-1362) to DN.

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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Fig. S1. Phylogenetic tree visualizing relationships between bacteria isolated during the sea spray experiments. Comparison of bacterial isolates and environmental clones from seawater (black) and aerosols (blue), from the total of 149 bacterial OTUs. Isolates are indicated by Latin name or major group affiliation and the code SVA preceding isolate number. Sequences cloned from seawater or aerosols are indicated by ‘Clone’ and the code SV preceding OTU number. Brackets indicate clusters containing bacterial isolates and/or clones with identical 16S rRNA gene sequences from both aerosols and seawater. Numbers at nodes denote values from 100 bootstrap replications. Scale bar depicts 0.05 substitutions per nucleotide position. Fig. S2. Phylogenetic tree of phytoplankton OTUs derived from chloroplasts (CL), mitochondria (ML) and cyanobacteria. Numbers at nodes denote values from 100 bootstrap replications. Scale bar depicts 0.05 substitutions per nucleotide position. Table S1. Summary of 16S rRNA gene clone library data generated from water collected from the three stations in Kongsfjorden, Svalbard. Values are for experimentally generated aerosols (Air) and the corresponding seawater (SW).

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports