<figure><img src="image_1.png" /></figure>

HAL
open science

# Effects of Saharan dust on the microbial community during a large in situ mesocosm experiment in the NW Mediterranean Sea

M. Laghdass, S. Blain, M. Besseling, P. Catala, C. Guieu, I. Obernosterer

► To cite this version:

M. Laghdass, S. Blain, M. Besseling, P. Catala, C. Guieu, et al. Effects of Saharan dust on the microbial community during a large in situ mesocosm experiment in the NW Mediterranean Sea. Aquatic Microbial Ecology, 2011, 62 (2), pp.201-213. 10.3354/ame01466. hal-03502043

HAL Id: hal-03502043

https://hal.science/hal-03502043

Submitted on 24 Dec 2021

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Effects of Saharan dust on the microbial community during a large *in situ* mesocosm experiment in the NW Mediterranean Sea

M. Laghdass<fnref n="1" />,<fnref n="2" />, S. Blain<fnref n="1" />,<fnref n="2" />, M. Besseling<fnref n="1" />,<fnref n="2" />, P. Catala<fnref n="1" />,<fnref n="2" />, C. Guieu<fnref n="3" />,<fnref n="4" />,
I. Obernosterer<fnref n="1" />,<fnref n="2" />,*

ABSTRACT: The response of the microbial community to Saharan dust deposition was investigated in 6 large mesocosms (52 m<fnref n="3" />) deployed at an oligotrophic coastal site in the NW Mediterranean Sea in June 2008 (DUNE project). The mesocosms represented well the environmental conditions observed at the study site during the 8 d experimental period, and the triplicate mesocosms exhibited high reproducibility for each treatment. Dust deposition resulted in an increase in chlorophyll a concentration (0.22 ± 0.03 µg l⁻<fnref n="1" />), as compared to that in the control treatments (0.12 ± 0.01 µg l⁻<fnref n="1" />), but no treatment effect was observed for bacterial heterotrophic abundance at 5 m depth. Results from the fingerprinting technique CE-SSCP indicate a temporal evolution of the structure of the total (16S rRNA gene) and active (16S rRNA transcripts) bacterial community, and Saharan dust deposition had a noticeable structuring effect on the active bacterial community. Combining results from 16S rRNA gene clone libraries and CE-SSCP indicates that the relative contribution of *Alteromonas macleodii* to the active bacterial community was enhanced 2-fold following dust addition. The 2 operational taxonomic units (OTUs) *Thiothrix* and *Alteromonas*, belonging to *Gammaproteobacteria*, and the *Bacteroidetes* OTU NS5-1 were specific to the clone libraries from the dust-amended mesocosms or more abundant in these than in the control ones. CARD-FISH analyses, however, indicate that these OTUs had overall low abundances (1 to 5% of total DAPI-counts). Despite the pronounced temporal trend observed during the experimental period, dust deposition had a small, but noticeable structuring effect on the heterotrophic bacterial community that was detectable only at the OTU level at 99% similarity of the 16S rRNA gene.

KEY WORDS: Saharan dust deposition · *In situ* mesocosms · CE-SSCP · CARD-FISH · Clone libraries · *Alteromonas macleodii* · NW Mediterranean Sea

Resale or republication not permitted without written consent of the publisher

## INTRODUCTION

Dry and wet atmospheric deposition represents a significant source of inorganic and organic nutrients and organic carbon to the ocean (Jickells et al. 2005; Jurado et al. 2008). In high-nutrient low-chlorophyll regions, dust deposition enhances iron availability and

thus stimulates phytoplankton primary production and potentially carbon dioxide drawdown (Boyd et al. 2007, Cassar et al. 2007). In low-nutrient low-chlorophyll (LNLC) regions, the simultaneous input of phosphate and iron from dust could create a favourable environment for enhanced dinitrogen fixation (Mills et al. 2004). In the subtropical northeast Atlantic Ocean,

<footnote n="1">¹UPMC Univ Paris 06, UMR 7621, LOMIC, Observatoire Océanologique, 66651 Banyuls/Mer, France</footnote>

<footnote n="2">²CNRS, UMR 7621, LOMIC, Observatoire Océanologique, 66651, Banyuls/Mer, France</footnote>

<footnote n="3">³UPMC Univ Paris 06, UMR 7093, LOV, Observatoire Océanologique, 06230 Villefranche/Mer, France</footnote>

<footnote n="4">⁴CNRS, UMR 7093, LOV, Observatoire Océanologique, 06230 Villefranche/Mer, France</footnote>

<footnote>*Corresponding author. Email: ingrid.obernosterer@obs-banyuls.fr</footnote>
dust addition markedly stimulated phytoplankton pri-
mary production and induced a shift from *Cyanobacte-
ria* to diatoms, while the response in microbial commu-
nity respiration was minor, resulting in an increase in
the ratio of primary production to respiration (Duarte
et al. 2006). In contrast, estuarine heterotrophic bacte-
rial and phytoplankton primary production were both
stimulated by organic nitrogen introduced by rainfall
(Seitzinger & Sanders 1999). These studies demon-
strate that atmospheric inputs can impact autotrophic
and heterotrophic microbial communities with impor-
tant consequences for ocean biogeochemistry.

In the Mediterranean Sea, which is during the strati-
fied period mostly a LNLC region, Saharan dust events
represent a dominant source of atmospheric deposi-
tion. Previous studies have demonstrated that, during
the summer stratification period, atmospheric dust is
the main external source of phosphorus (Ridame &
Guieu 2002) and of iron (Bonnet & Guieu 2006) in the
surface mixed layer. Microcosm experiments per-
formed in coastal and offshore Mediterranean waters
have shown that dust stimulated primary production
(Bonnet et al. 2005, Herut et al. 2005, Lekunberri et al.
2010) and is suspected to stimulate nitrogen fixation
(Bonnet & Guieu 2006). Direct observations of natural
dust deposition events in the eastern (Herut et al. 2005)
and western (Pulido-Villena et al. 2008) Mediter-
ranean Sea and in a Mediterranean reservoir (Reche et
al. 2009) revealed increases in the abundance and
metabolism of autotrophic and heterotrophic microbial
populations.

While the observed response of the phytoplankton
community was linked in some studies to changes in
major functional groups (Herut et al. 2005, Duarte et al.
2006), little is known about the effect of dust addition
on the structure of the heterotrophic bacterial com-
munity. Using microcosm experiments with water from
a Mediterranean reservoir, Reche et al. (2009) observed
no noticeable differences in the bacterial diversity and
richness between dust-amended and control treat-
ments. Conversely, dust addition induced changes in
the bacterial community composition in microcosms
performed with coastal Mediterranean waters (Lekun-
berri et al. 2010). The only study that has thus far inves-
tigated the impact of dust on the activity of specific bac-
terial groups reports that instantaneous metabolic rates
of *Prochlorococcus* and a bacterial subpopulation dom-
inated by SAR11 were reduced upon dust addition in
the North Atlantic Ocean (Hill et al. 2010). This study
suggests that the community response was linked to
changes in the microbial community structure (Hill et
al. 2010). The question of whether the response of spe-
cific bacterial groups to dust addition could play an im-
portant role in the cycling of elements introduced by
dust, however, remains poorly understood.

The objective of the present study was to investigate
the impact of dust deposition on the structure of the to-
tal and active bacterial community and to identify the
bacterial groups that were most responsive to dust ad-
dition. We used, for the first time in this context, large
trace-metal-clean *in situ* mesocosms that allowed us to
test for dust deposition effects close to *in situ* condi-
tions in LNLC waters.

MATERIALS AND METHODS

**Study site and sample collection.** The results pre-
sented here were obtained during the first field
campaign of the DUNE project (A DUst experiment
in a low Nutrient, low chlorophyll Ecosystem, www.
obs-vlfr.fr/LOV/DUNE). A detailed description of the
mesocosms, the collection and treatment of the Saha-
ran dust, the dust deposition and the sampling is given
by Guieu et al. (2010). Briefly, Saharan dust was col-
lected in Tunisia, a dominant source region of atmos-
pheric dust for the NW Mediterranean Sea. Saharan
dust was submitted to mechanical and physicochemi-
cal transformations before deposition in the meso-
cosms. The finest fraction (<20 µm) was used for the
experiment since dust particles larger than 20 µm are
rapidly removed during atmospheric transport (Maring
et al. 2003). In the present study, the fertilization mim-
icked a wet deposition event using evapocondensed
dust. Six large mesocosms, each containing 52 m³ of
raw seawater and extending to 15 m depth, were
deployed at a coastal site in the NW Mediterranean
Sea (Bay of Elbo, Corsica, France: 42° 22′ N, 8° 33′ E) in
June 2008. Of the 6 mesocosms, 3 were amended with
each 10 g m⁻² of Saharan dust simulating a relatively
strong atmospheric dust event (hereafter referred to as
D-mesocosms), and 3 mesocosms were established as
controls (hereafter referred to as C-mesocosms). Sam-
ples from the 6 mesocosms were taken at 5 m depth
before dust addition (t₀), and after 3 d (t₃), 6 d (t₆) and
8 d (t₈) after the amendment. Real-time measurements
revealed that several biogeochemical parameters
remained stable between t₃ and t₈; therefore, the
experiment was terminated after 8 d. Samples were
collected from a floating platform, using a Teflon
pump. Throughout the experiment, seawater was
also collected outside the mesocosms (hereafter re-
ferred to as OUT, n = 1) at a distance of about 50 m.
Water temperature was 19.8 ± 0.5°C inside all meso-
cosms throughout the experiment, as was the water
temperature outside the mesocosms.

**Enumeration of heterotrophic bacteria, autotrophic prokaryotic and eukaryotic cells.** The abundance of heterotrophic bacteria, autotrophic prokaryotic and eukaryotic cells was determined by flow cytometry
according to previously described protocols (Lebaron et al. 1998, Marie et al. 2000). Subsamples (2 ml for heterotrophic bacteria and 4 ml for autotrophic cells) were fixed with 2% formaldehyde (final conc.), incubated for 30 min at 4°C, frozen in liquid nitrogen and stored at -80°C until analysis. *Synechococcus* spp. were discriminated by their strong orange fluorescence (>630 nm) and pico- and nanoeukaryotes were discriminated by their scatter signals of the red fluorescence at 585 ± 21 nm. For heterotrophic bacteria, samples were stained with SYBR Green I (Invitrogen Molecular Probes) at 0.025% (vol/vol) final concentration for 15 min at room temperature in the dark. Counts were performed with a FACSCalibur flow cytometer (Becton Dickinson) equipped with an air-cooled argon laser (488 nm, 15 mW). Stained bacterial cells, excited at 488 nm, were discriminated and enumerated according to their right-angle light scatter (SSC, related to cell size) and green fluorescence measured at 530 ± 30 nm.

**Concentration of chlorophyll a.** To determine the concentration of chlorophyll a, 2 l of raw seawater were filtered on GFF filters (25 mm diameter). Filters were extracted in acetone, and the concentration of chlorophyll a was determined by fluorometry on a Trilogy Laboratory Fluorometer (Turner Designs).

**Nucleic acid extraction.** To determine the bacterial community structure by the fingerprinting method CE-SSCP, raw seawater (0.5–1 l) was first prefiltered through a 3 µm pore size filter (47 mm, Nucleopore), and the <3µm filtrate sequentially filtered through a 0.8 µm PC filter (47 mm, Nucleopore), and a 0.2 µm PC filter, respectively, using a peristaltic pump. The 0.8 µm and 0.2 µm PC filtrates were stored frozen at -80°C until DNA and RNA extraction. For both size fractions, the structure of the total (16S rRNA genes) and active (16S rRNA transcripts) bacterial communities was determined, based on the DNA and RNA extracts, respectively.

For DNA and RNA extraction the PC filters were submerged in 425 µl lysis buffer (40 mM EDTA, 50 mM Tris, 0.75 M sucrose) and then subjected to 3 freeze-thaw cycles using liquid nitrogen and a water bath at 65°C. Freshly prepared lysozyme solution (1 mg ml⁻¹) was added to the PC filters and incubated at 37°C for 45 min. Proteinase K (0.2 mg ml⁻¹) and sodium dodecyl sulphate (SDS 1%) were then added and the PC filters were incubated at 55°C for 1 h. Purification of DNA and RNA was performed with the Qiagen AllPrep DNA/RNA extraction kit. 1.5 ml RLT+ buffer (Qiagen) containing 14.3 M β-mercaptoethanol was added, mixed well and incubated for 10 min at room temperature. The DNA and RNA were purified separately from the lysate on the individual columns according to the manufacturer's instructions. The molecular size and

the purity of the DNA were analysed by agarose gel electrophoresis (1%).

**Reverse transcription of RNA.** RNA samples (2 µg) were incubated together with 0.5 ml primer w34 (1 mM final concentration) at 94°C for 5 min before cooling rapidly in a water/ice bath. Reverse transcription was achieved by adding 5 ml of deoxynucleoside triphosphates (dNTPs; 0.8 mM final concentration), 1 ml of Moloney murine leukemia virus (M-MLV) reverse transcriptase (20 U) and 5 µl of 5X M-MLV buffer (Promega) and allowing the reaction to proceed at 42°C for 1 h. The cDNA samples were stored at -20°C.

**Capillary electrophoresis – single-strand conformation polymorphism analysis (CE-SSCP).** CE-SSCP is a fingerprinting method that allows an image to be obtained of the structure of the total (16S rRNA genes) and active (16S rRNA transcripts) bacterial community. CE-SSCP enables determination of the presence and relative abundance of up to 40 of the most abundant operational taxonomic units (OTUs) of varying taxonomic ranks (Lami et al. 2009, West et al. 2008). Short fragments (~200 bp) of the V3 region of the 16S rRNA gene were amplified from DNA or from a cDNA copy of the RNA using the bacterial specific primers w49 (5'-ACG GTC CAG ACT CCT ACG GG-3') (Delbes et al. 2000) and w34 (5'-TTA CCG CGG TGC TGG CAC-3') (Lee et al. 1996), the latter 5' labelled with phosphoramidite (TET, Eurogentec). Amplifications were performed according to a previously described protocol (West et al. 2008). The electropherograms were analysed by GeneScan software (Applied Biosystems), using the second-order least square size calling method to normalize mobilities between different runs as described previously (Ghiglione et al. 2005). The similarity of the SSCP profiles was assessed as described previously (West et al. 2008) by using SAFUM software (Zemb et al. 2007), which normalizes the total area of the SSCP profiles and the mobilities between different runs using the internal standard. The Bray-Curtis distance was then calculated and similarity den-drograms were constructed using the complete linkage method by using Primer-E5 software package (Clarke & Warwick 2001).

**Assignment of environmental CE-SSCP peaks.** The 16S rRNA genes of several clones from each OTU determined from the clone libraries as described below were amplified directly from *E. coli* cells in PCR reaction mixes according to a previously described protocol (West et al. 2008). The clone electropherograms were superimposed on the environmental profiles for peak assignment. The areas of the peaks that could be assigned from the clones were obtained after peak detection by GeneScan Analysis.

**Sequencing and phylogenetic analysis of 16S rRNA gene clone libraries.** To investigate the composition of
the bacterial community, 16S rRNA clone libraries were constructed for the <0.8 µm fraction for one C-mesos cosm and one D-mesocosm at 5 m depth at the end of the experiment. The results from the clone libraries en- abled CE-SSCP peak assignment and probe design. The clone libraries were constructed according to the methods described previously (West et al. 2008). Plas- mid DNA (192 clones for each library) was sequenced using the BigDye™ Terminator kit and the 3730xl Automatic Sequencer by Macrogen using primer 27F (5'-AGA GTT TGA TCM TGG CTC AG-3'). The soft- ware BioEdit version 7.0.9.0 (Tom Hall, Ibis Therapeu- tics) was used to visualize the electropherograms and the sequences were trimmed by PHRED (Ewing et al. 1998). All sequences were checked for chimera forma- tion using CHECK_CHIMERA software (Maidak et al. 2001) and Bellerophon (Hugenholtzt & Huber 2003). A total of 33 clones were eliminated (plastids and bad se- quences). Phylogenetic groups or OTUs were defined at 99% similarity from the exported CLUSTALW align- ment using Clusterer software with the single linkage setting (Klepac-Ceraj et al. 2006). The closest relatives were found by submitting a representative sequence of each OTU to BLASTN. Each OTU was named by the closest BLAST hit or by clusters previously identified. One or more sequences from the OTU were fully se- quenced as described above using the primer 1492R (5'-TAC GGY TAC CTT GTT ACG ACT T-3') and the full-length sequences were assembled in BioEdit. The nucleotide sequences data from this study have been deposited in the GenBank database under the accession numbers HM117272 to HM117275 (plastid sequences), HM117276 to HM117458 (C-mesocosm bacterial sequences), HM117459 to HM117462 (cyano- bacterial sequences) and HM117263 to HM117626 (D-mesocosm bacterial sequences).

**Probe design and specificity.** The probes Thio- thrix_577a (5'-GCA TCA CAT CCT ACG TAC-3'), Thiothrix_577b (5'-GCA TTA CAT CCT ACG TAC-3') and NS5_175 (5'-TAT CAG GAT CGT ATG GGG-3') were designed and their specificity evaluated using the PROBE Design and PROBE Match tools of the ARB software package and the greengenes.arb database (Ludwig et al. 2004). As a cultured representative of the Thiothrix and NS5-1 OTU was not available, the hybridization conditions were optimized by carrying out hybridizations with the t~8~ samples using different formamide concentrations (from 45% to 60%). To obtain maximum specificity of the probe, the for- mamide concentration with highest fluorescence sig- nal was selected (55%).

**Abundance of major and specific bacteria using CARD-FISH.** We applied CARD-FISH (Pernthaler et al. 2002) to determine the quantitative importance of major phylogenetic and more specific bacterial groups.

CARD-FISH was performed using general and specific rRNA-targeted oligonucleotide probes labelled with horse-radish peroxidase (HRP; ThermoScientific). The probes Eub338-I, -II and -III (Amann et al. 1990, Daim et al. 1999), Alf968 (Amann et al. 1997), Gam42a (Manz et al. 1992) Bet42a and CF319a (Manz et al. 1996) were used to target most of the domain Bacteria and the major bacterial groups. A negative probe (the Eub antisense probe Non338) was used to determine non- specific binding (Glöckner et al. 1999). Unlabeled com- petitor probes were used for Betaproteobacteria and Gammaproteobacteria (Manz et al. 1992). The more specific probes used in this study were SAR11-152R, SAR11-441R, SAR11-542R, SAR11-732R (SAR11 clus- ter) (Morris et al. 2002), Ros537 (Roseobacter-Sulfito- bacter-Silicibacter group) (Eilers et al. 2001), SAR86 (SAR86 cluster) (Eilers et al. 2000) and ALT1413 (Alteromonas group) (Eilers et al. 2000). The recently designed probe NorSea72_88 (5'-GCC ACT CGT CAT CAG ATT-3') and 2 helper probes NorSea72_69H (5'- CCA AGC AAT CCA TGT TAC-3' and NorSea72_ 107H (5'-GCG TTA CGC ACC CGT GC-3') (NorSea72 group belonging to Bacteroidetes) (Laghdass et al. 2010) were also applied in the present study. Two probes were specifically designed to target the group Thiothrix belonging to Gammaproteobacteria (Thio- thrix_577a and Thiothrix_577b) and a part of the NS5 cluster (NS5-1) within Bacteroidetes (NS5_175) (see Table 2).

For each sample, 100 ml of raw seawater were filtered onto 0.2 µm PC membranes (47 mm) and incu- bated overnight at 4°C on pads soaked with 2% paraformaldehyde (PFA). Membranes were rinsed with 0.2 µm filtered Milli-Q water and dried before storing at -20°C until CARD-FISH analysis as described previ- ously (Pernthaler et al. 2002, Sekar et al. 2003). The to- tal number of cells (DAPI cells) and cells affiliated with a specific bacterial group (FITC labelled) were counted using semi-automated microscopy (Olympus Provis AX70) and an image analysis system (ImagePro). Data were collected from 10 counted fields with at least 300 cells per sample. Relative abundances of the bacte- rial groups are presented either as mean values and standard errors of cell counts of 10 microscopic fields or as mean values and standard deviations of triplicate filter pieces (see Table 3).

## RESULTS

### Response in the abundance and biomass of the microbial community

The addition of Saharan dust resulted in a rapid increase in phytoplankton biomass at 5 m depth
<table><caption>Table 1. Temporal changes in the chl *a* concentration (µg l⁻¹), the abundance of *Synechococcus* spp., pico- and nanoeukaryotes and heterotrophic bacteria at 5 m depth in the control mesocosms (C, n = 3), the dust-amended mesocosms (D, n = 3), and samples collected outside the mesocosms (OUT, n = 1). Samples were collected before dust-addition (t₀), after 3 d (t₃), 6 d (t₆) and 8 d (t₈). Mean values ± SD of the 3 C- and 3 D-mesocosms are given. At t₀, the samples of the C- and D-mesocosms, and the OUT sample were pooled (n = 7)</caption><thead><tr><th rowspan="2">Treatment</th><th rowspan="2">t<sub>0</sub></th><th colspan="3">t<sub>3</sub></th><th colspan="3">t<sub>6</sub></th><th colspan="3">t<sub>8</sub></th></tr><tr><th>C</th><th>D</th><th>OUT</th><th>C</th><th>D</th><th>OUT</th><th>C</th><th>D</th><th>OUT</th></tr></thead><tbody><tr><td>Chl <i>a</i></td><td>0.09 ± 0.02</td><td>0.13 ± 0.03</td><td>0.20 ± 0.01</td><td>0.12</td><td>0.09 ± 0.01</td><td>0.21 ± 0.03</td><td>0.10</td><td>0.12 ± 0.01</td><td>0.22 ± 0.03</td><td>0.14</td></tr><tr><td><i>Synechococcus</i> spp. (×10<sup>3</sup> cells ml<sup>-1</sup>)</td><td>14.6 ± 1.3</td><td>12.8 ± 1.9</td><td>16.8 ± 1.2</td><td>14.0</td><td>13.6 ± 3.9</td><td>16.8 ± 0.7</td><td>12.7</td><td>12.8 ± 1.1</td><td>16.6 ± 2.6</td><td>15.6</td></tr><tr><td>Pico- and nanoeukaryotes (cells ml<sup>-1</sup>)</td><td>471 ± 120</td><td>706 ± 192</td><td>805 ± 109</td><td>684</td><td>827 ± 197</td><td>1190 ± 73</td><td>645</td><td>946 ± 41</td><td>1120 ± 122</td><td>888</td></tr><tr><td>Heterotrophic bacteria (×10<sup>5</sup> cells ml<sup>-1</sup>)</td><td>3.40 ± 0.3</td><td>4.75 ± 0.5</td><td>5.13 ± 0.3</td><td>4.50</td><td>5.65 ± 0.2</td><td>4.75 ± 0.3</td><td>4.72</td><td>5.88 ± 0.3</td><td>5.25 ± 0.3</td><td>5.07</td></tr></tbody></table>

(Table 1). The initial concentration of chlorophyll *a* (chl *a*) was 0.09 µg l⁻¹ and it was about 2-fold higher in the D-mesocosms than in the C-mesocosms and the OUT samples throughout the experiment. The abundance of *Synechococcus* spp. was higher in the D-mesocosms (17 × 10³ cells ml⁻¹) than in the C-mesocosms and OUT samples (13 to 14 × 10³ cells ml⁻¹), but only at t₃ and t₆. Pico- and nanoeukaryotes revealed a different pattern, with higher abundances at t₆ and t₈ in the D-mesocosms (1155 ± 98 cells ml⁻¹, mean ± SD of t₆ and t₈, n = 6) than in the C-mesocosms (898 ± 121 cells ml⁻¹, mean ± SD of t₆ and t₈, n = 6) and the OUT samples (766 ± 171 cells ml⁻¹, mean ± SD of t₆ and t₈, n = 2) (Table 1). Before dust addition, heterotrophic bacterial abundance was 3.40 ± 0.3 × 10⁵ cells ml⁻¹ (mean ± SD, n = 7) and bacterial abundance varied between 4.5 and 5.88 × 10⁵ cells ml⁻¹ during the 8 d of the experiment with no pronounced differences detectable at 5 m depth between the D- and C-mesocosms (Table 1).

## Temporal pattern of the bacterial community structure

The structure of the total and active bacterial community was examined by 16S rRNA gene and the 16S rRNA transcript CE-SSCP profiles. The Bray-Curtis dendrograms based on the presence or absence of peaks (corresponding to OTUs) and on the relative peak height are presented in Fig. 1. Overall, the CE-SSCP profiles of the 3 replicate C-mesocosms and D-mesocosms revealed good reproducibility throughout the experiment. CE-SSCP profiles of the total bacterial community were clustered according to the time point of sampling and the size fraction, but independent of treatment (Fig. 1A). This pronounced temporal trend

was observed not only in all mesocosms, but also in the OUT samples. The dendrogram based on the rRNA transcripts, representative for the active bacterial community, revealed a similar temporal pattern (Fig. 1B). However, 8 d after dust addition, a treatment effect was detectable on the active community of the >0.8 µm size fraction (Fig. 1B).

## Composition of the bacterial community

To identify the most abundant OTUs of the bacterial communities, 16S rRNA gene clone libraries were constructed at t₈ for 1 C- and 1 D-mesocosm. Due to the higher number of OTUs present on the CE-SSCP profiles of the <0.8 µm size fraction (16 ± 1, mean ± SD, n = 6) than of the >0.8 µm size fraction (14 ± 0.5, mean ± SD, n = 5), the clone libraries were constructed for the former size fraction. A total of 351 clone sequences for the 2 clone libraries were analyzed. The cyanobacterial sequences accounted for 0.5 and 2% in the control and dust libraries, respectively, and they were excluded from further analyses. *Alphaproteobacteria* was the dominant group in both clone libraries (69 and 58% of total bacterial clone sequences in the C- and D-library, respectively) (Table 2). The contribution of *Bacteroidetes* was substantially smaller and similar in both clone libraries (about 10% of the total bacterial clone sequences). By contrast, the relative contribution of *Gammaproteobacteria* clones was roughly 2-fold higher in the D- than in the C-library (27 and 14% of total bacterial clone sequences, respectively).

When the sequences were clustered at 99% similarity of the 16S rRNA gene, a few noticeable differences between the 2 clone libraries were detectable. The cluster analysis resulted in the same number of OTUs
<table><caption>Table 2. Phylogenetic affiliation of each OTU in clone libraries from 1 control (C2) and 1 dust-amended (D2) mesocosm 8 d after dust addition (t8). Each OTU was named by the closest BLAST hit from GenBank or by clusters previously identified. The number of clones indicates the total number of clones determined in both libraries. For the control (C t8) and dust (D t8) libraries, the relative contribution of clones of major bacterial groups is presented as the percent of total clones of the respective treatment (in bold). The relative contribution of the OTUs is presented as the percent of clones of the respective major bacterial group. The phylogenetic clusters in grey represent clones with complete sequences, and the number of complete sequences is indicated in parentheses. Unc: uncultured</caption><thead><tr><th>Phylogenetic cluster</th><th>No. of clones</th><th>Representative clone</th><th>Closest relative</th><th>Accession Number</th><th>Identity (%)</th><th>Percent contribution C t8</th><th>D t8</th></tr></thead><tbody><tr><td><strong>Alphaproteobacteria</strong></td><td>221</td><td></td><td></td><td></td><td></td><td><strong>68.9</strong></td><td><strong>57.9</strong></td></tr><tr><td>SAR 11</td><td>161</td><td></td><td></td><td></td><td></td><td>76.2</td><td>68.4</td></tr><tr><td>SAR11-IA (3)</td><td>99</td><td>DUNE-P8C2-3</td><td>Unc. bacterium clone 2C228576</td><td>EU800465</td><td>99</td><td>46.8</td><td>42.1</td></tr><tr><td>SAR11-IIB (2)</td><td>46</td><td>DUNE-P8D2-125</td><td>Unc. marine bacterium clone SPOTSOCT00_5m28</td><td>DQ009168</td><td>99</td><td>20.6</td><td>21.1</td></tr><tr><td>SAR11-IB</td><td>3</td><td>DUNE-P8D2-188</td><td>Unc. marine bacterium clone SPOTSDEC01_5m31</td><td>DQ009250</td><td>99</td><td>0.0</td><td>3.2</td></tr><tr><td>SAR11-III</td><td>2</td><td>DUNE-P8C2-88</td><td>Unc. bacterium clone 6C232952</td><td>EU804972</td><td>99</td><td>1.6</td><td>0.0</td></tr><tr><td>SAR11-I</td><td>2</td><td>DUNE-P8C2-44</td><td>Unc. bacterium clone 6C232739</td><td>EU804778</td><td>99</td><td>1.6</td><td>0.0</td></tr><tr><td>Singletons</td><td>9</td><td></td><td></td><td></td><td></td><td>5.6</td><td>2.1</td></tr><tr><td><strong>Rhodobacterales</strong></td><td>7</td><td></td><td></td><td></td><td></td><td>2.4</td><td>4.2</td></tr><tr><td><strong>Rhodobacteraceae</strong></td><td>2</td><td>DUNE-P8C2-129</td><td>Marine metagenome 1096626661948</td><td>AACY020497968</td><td>99</td><td>0.8</td><td>1.1</td></tr><tr><td><strong>Rhodobacterales clade OCT</strong></td><td>3</td><td>DUNE-P8D2-26</td><td>Unc. bacterium clone 1C226901</td><td>EU799319</td><td>99</td><td>0.0</td><td>3.2</td></tr><tr><td>Singletons</td><td>2</td><td></td><td></td><td></td><td></td><td>1.6</td><td>0.0</td></tr><tr><td>Other</td><td>53</td><td></td><td></td><td></td><td></td><td>21.4</td><td>27.4</td></tr><tr><td><strong>Aegean 169 (2)</strong></td><td>8</td><td>DUNE-P8D2-21</td><td>Unc. marine bacterium clone SPOTSAUG01_5m781</td><td>DQ009261</td><td>99</td><td>4.0</td><td>3.2</td></tr><tr><td><strong>OCS 116 clade (2)</strong></td><td>9</td><td>DUNE-P8C2-143</td><td>Unc. bacterium clone ZA3318c</td><td>AF382114</td><td>99</td><td>3.2</td><td>5.3</td></tr><tr><td>SAR116</td><td>2</td><td>DUNE-P8C2-188</td><td>Unc. marine bacterium clone SPOTSAUG01</td><td>DQ009267</td><td>99</td><td>1.6</td><td>0.0</td></tr><tr><td><strong>Rickettsiales</strong></td><td>2</td><td>DUNE-P8C2-152</td><td>Unc. bacterium clone 1C227245</td><td>EU799623</td><td>97</td><td>1.6</td><td>0.0</td></tr><tr><td>SAR116-2</td><td>7</td><td>DUNE-P8D2-17</td><td>Unc. alphaproteobacterium CHAB-III-8</td><td>A1240913</td><td>99</td><td>2.4</td><td>4.2</td></tr><tr><td>SAR116-3</td><td>4</td><td>DUNE-P8C2-141</td><td>Unc. marine bacterium clone SPOTSAUG01_5m101</td><td>DQ009267</td><td>99</td><td>2.4</td><td>1.1</td></tr><tr><td>Aegean 169-2</td><td>4</td><td>DUNE-P8D2-34</td><td>Unc. marine bacterium clone SPOTSAUG01_5m94</td><td>DQ009262</td><td>99</td><td>1.6</td><td>2.1</td></tr><tr><td>SAR116-3</td><td>3</td><td>DUNE-P8D2-39</td><td>Unc. marine bacterium clone SPOTSAUG01_5m94</td><td>DQ009276</td><td>99</td><td>0.0</td><td>3.2</td></tr><tr><td>SAR116-4</td><td>4</td><td>DUNE-P8D2-39</td><td>Unc. marine bacterium clone 1C227519</td><td>EU799856</td><td>99</td><td>0.8</td><td>3.2</td></tr><tr><td>Singletons</td><td>10</td><td></td><td></td><td></td><td></td><td>4.0</td><td>5.3</td></tr><tr><td><strong>Betaproteobacteria</strong></td><td>3</td><td></td><td></td><td></td><td></td><td><strong>1.1</strong></td><td><strong>0.6</strong></td></tr><tr><td>OM43</td><td>2</td><td>DUNE-P8C2-179</td><td>Unc. bacterium clone 2C228624</td><td>EU800503</td><td>99</td><td>100</td><td>0.0</td></tr><tr><td>Singletons</td><td>1</td><td></td><td></td><td></td><td></td><td>0.0</td><td>100</td></tr><tr><td><strong>Gammaproteobacteria</strong></td><td>69</td><td></td><td></td><td></td><td></td><td><strong>13.7</strong></td><td><strong>26.8</strong></td></tr><tr><td><strong>Alteromonas macleodii (6)</strong></td><td>15</td><td>DUNE-P8D2-1</td><td>Alteromonas sp. MOLA-78, culture collection MOLA 78.</td><td>AM990853</td><td>99</td><td>20.0</td><td>22.7</td></tr><tr><td><strong>JTB148 (2)</strong></td><td>12</td><td>DUNE-P8D2-3</td><td>Unc. gammaproteobacterium clone SHWH_night1_16S_456</td><td>FJ745010</td><td>98</td><td>20.0</td><td>15.9</td></tr><tr><td>SAR86-1</td><td>6</td><td>DUNE-P8D2-163</td><td>Marine metagenome ctg. 1101668521133</td><td>AACY023713782</td><td>99</td><td>20.0</td><td>2.3</td></tr><tr><td>NAC11-19</td><td>7</td><td>DUNE-P8C2-187</td><td>Unc. gammaproteobacterium NAC11-19</td><td></td><td>99</td><td>12.0</td><td>9.1</td></tr><tr><td>Vibrio</td><td>4</td><td>DUNE-P8D2-30</td><td>Vibrio sp. S1346</td><td>FJ457385</td><td>99</td><td>4.0</td><td>6.8</td></tr><tr><td><strong>Thiobrix (2)</strong></td><td>5</td><td>DUNE-P8D2-36</td><td>Unc. bacterium clone 1C227670</td><td>EU799990</td><td>98</td><td>0.0</td><td>11.4</td></tr><tr><td>SAR86-2</td><td>2</td><td>DUNE-P8C2-9</td><td>Unc. gammaproteobacterium clone PEACE2006/149_P2</td><td>EU394552</td><td>99</td><td>4.0</td><td>2.3</td></tr><tr><td>K189A</td><td>3</td><td>DUNE-P8C2-183</td><td>Unc. bacterium clone S25_881</td><td>EF574537</td><td>99</td><td>8.0</td><td>2.3</td></tr><tr><td><strong>Pseudoalteromonas</strong></td><td>2</td><td>DUNE-P8C2-45</td><td>Pseudoalteromonas sp. UST020129-030</td><td>EU982330</td><td>99</td><td>4.0</td><td>2.3</td></tr><tr><td>SAR86-3</td><td>2</td><td>DUNE-P8D2-52</td><td>Unc. bacterium clone S25_909</td><td>EF574565</td><td>99</td><td>0.0</td><td>4.5</td></tr><tr><td>Singletons SAR 86</td><td>3</td><td></td><td></td><td></td><td></td><td>4.0</td><td>4.5</td></tr><tr><td>Singletons other</td><td>8</td><td></td><td></td><td></td><td></td><td>4.0</td><td>15.9</td></tr></tbody></table>
<table><caption>Table 2 (continued)</caption><thead><tr><th>Phylogenetic cluster</th><th>No. of clones</th><th>Representative clone</th><th>Closest relative</th><th>Accession Number</th><th>Identity (%)</th><th>Percent contribution C t<sub>8</sub></th><th>Percent contribution D t<sub>8</sub></th></tr></thead><tbody><tr><td><strong>Bacteroidetes</strong></td><td></td><td></td><td></td><td></td><td></td><td><strong>10.9</strong></td><td><strong>8.5</strong></td></tr><tr><td>NS5-1 (2)</td><td>34</td><td>DUNE-P8C2-120</td><td>Unc. Flavobacteria bacterium clone NorSea37</td><td>AM279169</td><td>98</td><td>5.0</td><td>28.6</td></tr><tr><td>NS4 (NorSea72)</td><td>5</td><td>DUNE-P8C2-144</td><td>Unc. Flavobacteria bacterium clone NorSea72</td><td>AM279212</td><td>98</td><td>15.0</td><td>7.1</td></tr><tr><td>NS4-1</td><td>4</td><td>DUNE-P8D2-181</td><td>Unc. bacterium clone 6C233366</td><td>EU805360</td><td>97</td><td>0.0</td><td>14.3</td></tr><tr><td>Aegean179</td><td>2</td><td>DUNE-P8C2-24</td><td>Unc. marine bacterium clone SPOTSAUG01_5m55</td><td>DQ009099</td><td>97</td><td>10.0</td><td>0.0</td></tr><tr><td>NS5-2</td><td>2</td><td>DUNE-P8C2-135</td><td>Unc. Flavobacterium sp. clone SHWH-night1</td><td>FJ744962</td><td>93</td><td>10.0</td><td>0.0</td></tr><tr><td>NS7</td><td>2</td><td>DUNE-P8C2-189</td><td>Unc. Flavobacteria bacterium clone NorSea23</td><td>AM279183</td><td>99</td><td>10.0</td><td>0.0</td></tr><tr><td>NS4-2</td><td>2</td><td>DUNE-P8D2-69</td><td>Unc. Flavobacteria bacterium clone NorSea72</td><td>AM279212</td><td>96</td><td>5.0</td><td>7.1</td></tr><tr><td>Winogradskyella Singletons</td><td>2</td><td>DUNE-P8C2-71</td><td>Unc. Flavobacteriaceae bacteriumclone Sylt 16</td><td>AM040112</td><td>95</td><td>10.0</td><td>0.0</td></tr><tr><td></td><td>13</td><td></td><td></td><td></td><td></td><td>35.0</td><td>42.9</td></tr><tr><td><strong>Actinobacteria</strong></td><td>13</td><td></td><td></td><td></td><td></td><td><strong>3.3</strong></td><td><strong>4.3</strong></td></tr><tr><td>Actinobacteria 1</td><td>5</td><td>DUNE-P8D2-2</td><td>Unc. bacterium clone 6C232606</td><td>EU804655</td><td>99</td><td>33.3</td><td>42.9</td></tr><tr><td>Actinobacteria 2</td><td>6</td><td>DUNE-P8D2-41</td><td>Unc. marine bacterium clone SPOTSAUG01_5m99</td><td>DQ157868</td><td>99</td><td>50.0</td><td>42.9</td></tr><tr><td>Actinobacteria 3</td><td>2</td><td>DUNE-P8C2-162</td><td>Unc. marine bacterium clone SPOTSAUG01_5m99</td><td>AM748181</td><td>99</td><td>16.7</td><td>14.3</td></tr><tr><td><strong>Verrucomicrobia</strong></td><td></td><td></td><td></td><td></td><td></td><td><strong>2.2</strong></td><td><strong>1.2</strong></td></tr><tr><td>Verrucomicrobia</td><td>6</td><td>DUNE-P8C2-87</td><td>Unc. bacterium CHAB-II-49</td><td>AJ240909</td><td>99</td><td>25</td><td>50</td></tr><tr><td>Singletons</td><td>2</td><td></td><td></td><td></td><td></td><td>75</td><td>50</td></tr><tr><td></td><td>4</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Lentisphaerae lentisphaera</strong></td><td>1</td><td>DUNE-P8D2-147</td><td>Unc. bacterium clone 4C230431</td><td>EU803027</td><td>99</td><td>0.0</td><td>100</td></tr><tr><td>Singletons</td><td>1</td><td></td><td></td><td></td><td></td><td>0.0</td><td>100</td></tr></tbody></table>

(33) and singletons (26) for both libraries (Table 2). A higher number of OTUs was determined for *Alphaproteobacteria* (16 OTUs), whereas only 10 and 8 OTUs were determined for *Gammaproteobacteria* and *Bacteroidetes*, respectively (Fig. 2). Within *Alphaproteobacteria*, the 2 OTUs SAR11-IA and SAR11-IB (Kan et al. 2008) were the most dominant OTUs in both libraries (44 ± 3 and 21 ± 0.2% of *Alphaproteobacteria* clones, respectively, mean ± SD, n = 2). *Gammaproteobacteria* were dominated by the 3 OTUs *Alteromonas macleodii*, JTB148 and SAR86-1 in the C-library, each accounting for 20% of *Gammaproteobacteria* clones. By contrast, in the D-library, only the OTU *A. macleodii* had an important relative contribution (23% of *Gammaproteobacteria* clones). In the D-mesocosm, the number of total clones of *A. macleodii* (10) was 2-fold higher than in the C-mesocosm (5). It was further interesting to note that the OTU Thiothrix was detected only in the D-library, where it represented 11% of *Gammaproteobacteria* clones. Within the *Bacteroidetes*, the OTU NS4-1 was specific to the D-library (14% of *Bacteroidetes* clones) and the OTU NS5-1 had the highest relative contribution to *Bacteroidetes* clones in this library (28% of *Bacteroidetes* clones). In contrast, in the C-library the 4 OTUs NS5-2, Aegean179, NS7 and Winogradskyella contributed equally to *Bacteroidetes* (each accounting for roughly 10% of *Bacteroidetes* clone sequences). We observed no differences in the contribution of the 3 OTUs determined within the *Actinobacteria* between the 2 clone libraries.

A *J*-LIBSHUFF analysis (Schloss et al. 2004) allowed comparison of the 2 clone libraries. When the homologous coverage curve of one library (X) was compared with the heterologous curve XY, neither the comparison $(C_X-C_{XY})^2$ nor the reverse comparison $(C_Y-C_{YX})^2$ showed a significant difference between the 2 libraries, with values of $p = 0.0631$ and $p = 0.1928$ for $(C_X-C_{XY})^2$ and $(C_Y-C_{YX})^2$, respectively.

### Identification of specific CE-SSCP peaks

To identify the CE-SSCP peaks that were different between the C- and the D-mesocosms at $t_8$ we performed CE-SSCP analysis of several clones from each OTU identified, and
<figure><img src="image_3.png" /><figcaption>Fig. 1. Similarity dendrograms of the CE-SSCP profiles of (A) the 16S rRNA gene and (B) the 16 rRNA transcripts of the bacterial communities based on the Bray-Curtis distances. The dendrogram was constructed by the complete linkage method. t₀, t₃, and t₈ refer to sample collection before dust addition, and 3 d and 8 d after dust addition, respectively. D1, D2, D3: dust-amended mesocosms; C1, C2, C3: control mesocosms; OUT: samples collected outside the mesocosms; 0.2 µm: <0.8 µm size fraction; 0.8 µm: 0.8–3 µm size fraction; DNA: CE-SSCP profiles based on 16S rRNA gene; RNA: CE-SSCP profiles based on 16S rRNA transcript</figcaption></figure>

aligned the migration of the clones to the environmental
community profile (Fig. 2). One peak that revealed no-
ticeable differences between the C- and the D-meso-
cosms was identified as *Alteromonas macleodii* at the
end of the experiment (Fig. 2A,B). For the 16S rRNA
transcript-based profile of the >0.8 µm size fraction, the
relative contribution of this peak to total peak area was
about 2-fold higher in the D-mesocosms than that in the
C-mesocosms (14 ± 8 and 7 ± 1% of the total peak area,
respectively, n = 3). For the 16S rRNA gene based profile,
the relative contribution of this peak to total peak area
was 11 ± 2 and 16 ± 6% (n = 3) for the C- and D-meso-
cosms, respectively. For the <0.8 µm size fraction, the
relative contribution of this peak to total peak area was,

respectively, 2 ± 1 and 4 ± 2% for the C- and D-meso-
cosms for the 16S rRNA gene-based profile, and, respec-
tively, 9 ± 1 and 6 ± 6% for the C- and D-mesocosms for
the 16S rRNA transcript-based profile. Other peaks iden-
tified were SAR86, SAR11 and *Synechococcus*, but no
differences were detectable for these OTUs.

**Contribution of bacterial groups to total abundance**

We used previously designed and 2 specifically-
designed probes for the OTUs Thiothrix and NS5-1
(See ‘Materials and methods’) to quantify the relative
abundance of the major and some more specific bacte-
<figure><img src="image_4.png" /><figcaption>Fig. 2. (A) 16S rRNA gene and (B) 16S rRNA transcript CE-SSCP profiles showing different peak patterns for the 0.8–3 µm size fraction in 1 representative control mesocosm (C1, solid line) superimposed on 1 dust-amended mesocosm (D1, broken line). The shaded stripes show the positions of peaks that were assigned by the clones (named below). SAR86 is a tentatively assigned peak</figcaption></figure>

<table><caption>Table 3. Relative contribution of bacterial groups to bulk bacterial abundance before (t₀) and 8 d after dust deposition (t₈) in control (C) and dust-amended (D) mesocosms, and in samples collected outside the mesocosms (OUT). Means ± SD of replicate mesocosms are given, except for the OUT sample (t₈ OUT) where mean values ± SE of 10 microscopic fields are given. For t₀, results from the 3 C- and D-mesocosms, and the OUT sample are pooled</caption><thead><tr><th rowspan="3"></th><th colspan="4">Relative contribution of bacterial groups<br>(% DAPI-cells)</th></tr><tr><th>t<sub>0</sub><br>(n = 7)</th><th>t<sub>8</sub><br>C (n = 3)</th><th>t<sub>8</sub><br>D (n = 3)</th><th>t<sub>8</sub><br>OUT (n = 1)</th></tr></thead><tbody><tr><td>Bacteria</td><td>67 ± 15</td><td>71 ± 10</td><td>62 ± 10</td><td>60 ± 3</td></tr><tr><td>Alphaproteobacteria</td><td>36 ± 10</td><td>32 ± 13</td><td>42 ± 1</td><td>31 ± 3</td></tr><tr><td>SAR11</td><td>31 ± 6</td><td>42 ± 7</td><td>37 ± 7</td><td>34 ± 2</td></tr><tr><td>Roseobacter</td><td>8 ± 3</td><td>9 ± 1</td><td>6 ± 1</td><td>9 ± 2</td></tr><tr><td>Betaproteobacteria</td><td>12 ± 4</td><td>14 ± 1</td><td>7 ± 1</td><td>10 ± 1</td></tr><tr><td>Gammaproteobacteria</td><td>23 ± 5</td><td>21 ± 4</td><td>20 ± 6</td><td>23 ± 2</td></tr><tr><td>SAR86</td><td>8 ± 2<sup>a</sup></td><td>7 ± 2</td><td>8 ± 3</td><td>8 ± 1</td></tr><tr><td>Alteromonas</td><td>2 ± 0.8<sup>a</sup></td><td>3 ± 1</td><td>5 ± 2</td><td>3 ± 2</td></tr><tr><td>Thiothrix</td><td>1 ± 0.3<sup>a</sup></td><td>2 ± 1</td><td>1 ± 0.1</td><td>2 ± 1</td></tr><tr><td>Bacteroidetes</td><td>34 ± 9</td><td>17 ± 4</td><td>34 ± 9</td><td>27 ± 3</td></tr><tr><td>NorSea72</td><td>4 ± 1<sup>a</sup></td><td>5 ± 0.3</td><td>5 ± 2</td><td>5 ± 1</td></tr><tr><td>NS5-1</td><td>0.7 ± 0.6<sup>a</sup></td><td>0.7 ± 0.1</td><td>2 ± 0.2</td><td>2 ± 0.4</td></tr></tbody><tfoot><tr><td colspan="5"><sup>a</sup>n = 3</td></tr></tfoot></table>

rial groups determined by the clone libraries. Overall, no differences in the abundance of the major bacterial groups were detectable between treatments, and we observed some minor differences only at the OTU level. For the description of the relative abundance of most of the bacterial groups determined in the present study, we have therefore pooled the results from the different treatments and time points (n = 14). The proportion of cells that hybridized with the general EUB probes and the NON338 probe was on average 65 ± 13% (n = 14) and 3 ± 2% (n = 14) respectively. SAR11 had important contributions to bulk abundance in the C- and D-mesocosms and in the OUT samples throughout the experiment (35 ± 7% of DAPI-stained cells respectively, n = 14, results from all treatments and time points pooled), while the contribution of *Roseobacter* remained low (8 ± 3% of DAPI-stained cells, n = 14) (Table 3). The relative contributions of *Gammaproteobacteria*, SAR86 and Thiothrix were independent of treatment and did not vary over time (22 ± 4, 8 ± 2, 1 ± 1% of DAPI-stained cells, respectively, n = 14). Even though relative abundances were overall low (2 to 5% of DAPI-stained cells), *Alteromonas* revealed slightly higher abundances in the D-than in the C-mesocosms 8 d after dust addition (5 and 3% of DAPI-stained cells, respectively, n = 3 in both cases). *Bacteroidetes* displayed similar relative abundances in the D-mesocosms and the OUT samples throughout the experiment (31 ± 6% of DAPI-stained cells, n = 11, data from all D-mesocosms and OUT samples pooled), but the relative abundance of *Bacteroidetes* was substantially lower in the C-mesocosms at t₈ (17 ± 4% of DAPI-stained cells, n = 3) (Table 3). While the OTU NorSea72 did not display any differences between treatments (5 ± 1% of DAPI-stained cells, n = 14), the OTU NS5-1 was slightly more abundant in the D-than in the C-mesocosms at t₈ (2 ± 0.2% and 0.7 ± 0.1% of DAPI-stained cells, respectively, n = 3 in both cases). Combining the results of all bacterial groups considered here, no significant differences between the C- and the D-mesocosms were detectable after 8 d (Kruskal-Wallis, p > 0.8).

DISCUSSION

The impact of dust deposition on the biogeochemistry of a LNLC marine ecosystem and in particular on bacterial community structure has not, to the best of our knowledge, so far been investi-
gated using trace-metal-clean *in situ* mesocosms. The 2
previous studies that addressed this question were car-
ried out in microcosm experiments (up to 15 l) (Reche et
al. 2009, Lekunberri et al. 2010). The lack of *in situ* obser-
vations is due to the episodic nature of atmospheric dust
deposition that makes it difficult to follow related *in situ*
changes at appropriate temporal and spatial scales. For
the biogeochemical parameters determined during the
DUNE project and the bacterial community structure-
the control mesocosms represented the temporal
changes that occurred in the Bay of Elbo well (Guieu et
al. 2010), and the 3 replicate mesocosms exhibited high
reproducibility in this study. Large *in situ* mesocosms
represent therefore an original and appropriate tool to
investigate an atmospheric dust event in a LNLC envi-
ronment in close to *in situ* conditions.

During the DUNE project, dust deposition resulted in
a rapid, but transient, increase in dissolved inorganic
phosphate concentrations by 12 nM at the surface
(Pulido-Villena et al. 2010). From our observations,
dust deposition resulted in an approximately 2-fold
increase in the concentration of chl a in the upper 5 m
over the duration of the experiment(Table 1). In con-
trast, bacterial heterotrophic abundance did not reveal
any marked increase at 5 m depth following dust addi-
tion (Table 1). The P-fertilizing effect of dust on the
autotrophic community confirms previous microcosm
experiments in the Mediterranean Sea (Bonnet et al.
2005, Herut et al. 2005, Lekunberri et al. 2010) and it
has also been demonstrated for heterotrophic bacterial
metabolism (Herut et al. 2005, Pulido-Villena et al.
2008, Reche et al. 2009, Lekunberri et al. 2010). Only in
one study was this bulk response accompanied by
noticeable changes in the bacterial community struc-
ture (Lekunberri et al. 2010). In this study, the addition
of dust or inorganic phosphorus (P) to coastal Mediter-
ranean waters resulted in a similar bacterial commu-
nity structure in both treatments. This observation led
to the conclusion that P introduced by dust addition
was the factor driving observed changes in bacterial
community structure (Lekunberri et al. 2010). Changes
in bacterial community structure following P addition
to Mediterranean waters are also reported from previ-
ous experimental studies (Lebaron et al. 2001, Schäfer
et al. 2001, Pinhassi et al. 2006). In these previous stud-
ies, the final P concentrations were, however, substan-
tially higher (0.3 to 0.6 µM) than in the DUNE meso-
cosms amended with dust (≤17 nM) (Pulido-Villena et
al. 2010). If, in the present study, the input of the low P
concentration due to dust addition had a structuring
effect on the bacterial community composition, it was
probably hidden in the natural temporal trend (Fig. 1).

Over the 8 d of the experiment, we observed a pro-
nounced temporal evolution of the total (16S rRNA
genes) and active (16S rRNA transcripts) bacterial

community structure (Fig. 1). Changes in bacterial
community structure are frequently observed on a sea-
sonal time scale (Ghiglione et al. 2005, Fuhrman et.
2006, Mary et al. 2006, Lami et al. 2009), but they can
also occur on shorter temporal scales (Hewson et al.
2006, Lamy et al. 2009). Changes in bacterial commu-
nity structure on a weekly time scale could be of
importance during different phases of phytoplankton
blooms, phases of transition between seasons or fol-
lowing environmental perturbations (Fandino et al.
2001, West et al. 2008). Roughly 2 wk prior to the
DUNE experiment, a Saharan dust event (K. Desboeuf
& F. Dulac pers. comm.) and heavy rainfalls (Wagener
et al. 2010) occurred at the study site, which could
explain the temporal pattern in bacterial community
structure we observed during our study.

Despite this natural temporal trend, dust deposition
had an impact, detectable at the OTU level, on a few
particular features of the bacterial community compo-
sition. The OTUs *Thiothrix* and *Alteromonas macleodii*, belonging to *Gammaproteobacteria*, and the *Bacteroidetes* OTU NS5-1 were more abundant in or
specific to the dust-amended mesocosms, compared to
the control mesocosms at the end of the experiment
(Table 2; Fig.2). The presence of these OTUs has so far
not been linked to dust deposition. The OTU *Thiothrix*
has been observed in clone libraries of the North
Pacific coast (Brown et al. 2005) and it was present in
many samples taken during the Global Ocean Sam-
pling (Shaw et al. 2008). The cluster NS5 appears to be
common in the coastal environment of temperate
(DeLong et al. 1993, Alonso et al. 2007) and high lati-
tude regions (Bano & Hollibaugh 2002). A noticeable
pattern observed in our study was the relatively high
contribution of the species *A. macleodii* to the active
bacterial community of the >0.8 µm fraction in the D-
mesocosms (Fig. 2). *A. macleodii* and related species
are among the most widespread cultured *Gammapro-
teobacteria*. Previous observations that *A. macleodii*
rapidly responds in nutrient enrichment experiments
(Pukall et al. 1999, Schäfer et al. 2000) and that its pres-
ence *in situ* is often found in association with particu-
late matter (Acinas et al. 1999, López-López et al. 2005)
have led to the idea that *A. macleodii* has an oppor-
tunistic way of life, which favours its dominance in
response to enhanced nutrient supply. The analysis
of the complete genome of a surface ecotype of *A.
macleodii* indicates that it is well adapted to rapidly
changing environmental conditions, by means of com-
plex patterns of regulation and environmental sensing,
features that could be linked to transient particle
attachment (Ivars-Martinez et al. 2008).

Beside the clear impact on P cycling mentioned
above, it is also expected that dust addition alters iron
cycling. During the DUNE experiment, dissolved iron
concentrations (Guieu et al. 2010, Wagener et al. 2010) decreased rapidly following dust addition, mainly due to scavenging by rapidly sinking large dust particles. This was confirmed by measurements of particulate iron and aluminium, and the analysis of the sediment traps deployed below each of the mesocosms that revealed that a fraction of dust particles rapidly sunk after their deposition at the surface (Guieu et al. 2010, Wagener et al. 2010). Based on Stokes's law, we estimated that dust particles with a diameter <3.2 µm remained in the upper 5 m layer of the D-mesocosms until the end of the experiment. These small particles represented 20% of the total volume of dust introduced into the mesocosms at the beginning of the experiment, and they could provide a new, but not directly available, source of iron to microorganisms. Grazing of these particles by protists could be one means of liberating colloidal- or particle-bound iron as dissolved iron (Barbeau et al. 1996). The reduced availability of dissolved iron and the presence of small iron-rich particles could also act jointly to stimulate the production of siderophores by heterotrophic bacteria (Granger & Price 1999, Kraemer 2004). We hypothesize that small Saharan dust particles could have provided a favourable environment for iron acquisition by heterotrophic bacteria and could explain the increased activity of some bacteria like *Alferomonas macleodii* in the D-mesocosms. Results from experiments conducted in parallel to this study demonstrate that the iron solubility capacity of seawater in the D-mesocosms increased by the end of the experiment (Wagener et al. 2010) and lend support to our hypothesis that dust addition stimulates the production of siderophores by heterotrophic bacteria.

*Acknowledgements.* The work presented here is part of the doctoral dissertation of M.L. supported by a grant of the French Ministère de l'Éducation Nationale de l'Enseignement Supérieur et de la Recherche. The DUNE project was funded by the ANR under the contract 'ANR-07-BLAN-0126-01'. We thank the 'Réserve naturelle de Scandola Parc Naturel Régional de Corse' in particular J.M. Dominici and collaborators for professionalism and cooperation in the implementation of the field work in the Bay of Elbo. We thank M. Deschates for her help in sample collection and filtration. The involvement of F. Louis, J. M. Grisoni, D. Luquet, C. Ridame, E. Pulido-Villena, C. Brunet, L. Gilleta and C. Rouvières during the DUNE-1 experiment is greatly acknowledged. We are grateful to N. West for her help in probe design and C. Manes for her advice on recovery of DNA and RNA from environmental samples. We thank 2 anonymous reviewers who helped to improve the manuscript.

## LITERATURE CITED

<figure><img src="image_5.png" /></figure>

* Acinas SG, Anton J, Rodriguez-Valera F (1999) Diversity of free-living and attached bacteria in offshore Western Mediterranean waters as depicted by analysis of genes encoding 16S rRNA. Appl Environ Microbiol 65:514–522

<figure><img src="image_6.png" /></figure>

* Alonso C, Warnecke F, Amann R, Pernthaler J (2007) High local and global diversity of *Flavobacteria* in marine plankton. Environ Microbiol 9:1253–1266

<figure><img src="image_7.png" /></figure>

* Amann RI, Krumholz L, Stahl DA (1990) Fluorescent-oligo-nucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 172:762–770

<figure><img src="image_8.png" /></figure>

* Amann R, Glockner F, Neef A (1997) Modern methods in subsurface microbiology: in situ identification of microorganisms with nucleic acid probes. FEMS Microbiol Rev 20:191–200

<figure><img src="image_9.png" /></figure>

* Bano N, Hollibaugh J (2002) Phylogenetic composition of bacterioplankton assemblages from the Arctic Ocean. Appl Environ Microbiol 68:505–518

<figure><img src="image_10.png" /></figure>

* Barbeau K, Moffett J, Caron D, Croot P, Erdner D (1996) Role of protozoan grazing in relieving iron limitation of phytoplankton. Nature 380:61–64

<figure><img src="image_11.png" /></figure>

* Bonnet S, Guieu C (2006) Atmospheric forcing on the annual iron cycle in the western Mediterranean Sea: a 1-year survey. J Geophys Res 111, C09010, doi:10.1029/2005JC003213

<figure><img src="image_12.png" /></figure>

* Bonnet S, Guieu C, Chiaverini J, Ras J, Stock A (2005) Effect of atmospheric nutrients on the autotrophic communities in a low nutrient, low chlorophyll system. Limnol Oceanogr 50:1810–1819

<figure><img src="image_13.png" /></figure>

* Boyd PW, Jickells T, Law CS, Blain S and others (2007) Mesoscale iron enrichment experiments 1993–2005: synthesis and future directions. Science 315:612–617

<figure><img src="image_14.png" /></figure>

* Brown MV, Schwalbach MS, Hewson I, Fuhrman JA (2005) Coupling 16S-ITS rDNA clone libraries and automated ribosomal intergenic spacer analysis to show marine microbial diversity: development and application to a time series. Environ Microbiol 7:1466–1479

<figure><img src="image_15.png" /></figure>

* Cassar N, Bender M, Barnett B, Fan S, Moxim W, Levy H, Tilbrook B (2007) The Southern Ocean biological response to aeolian iron deposition. Science 317:1067–1070

* Clarke K, Warwick R (2001) Changes in marine communities: an approach to statistical analysis and interpretation, 2nd ed. Primer-E, Plymouth

<figure><img src="image_16.png" /></figure>

* Daim H, Brühl A, Amann R, Schleifer KH, Wagner M (1999) The domain-specific probe EUB 338 is insufficient for the detection of all bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22: 434–444

<figure><img src="image_17.png" /></figure>

* Delbes C, Moletta R, Godon JJ (2000) Monitoring of activity dynamics of an anaerobic digester bacterial community using 16S rRNA polymerase chain reaction-single-strand conformation polymorphism analysis. Environ Microbiol 2:506–515

<figure><img src="image_18.png" /></figure>

* DeLong E, Franks D, Alldredge A (1993) Phylogenetic diversity of aggregate-attached vs. free-living marine bacterial assemblages. Limnol Oceanogr 38:924–934

<figure><img src="image_19.png" /></figure>

* Duarte C, Dachs J, Llabrés M, Alonso-Laita P and others (2006) Aerosol inputs enhance new production in the subtropical northeast Atlantic. J Geophys Res 111, G04006, doi:10.1029/2005JG000140

<figure><img src="image_20.png" /></figure>

* Eilers H, Pernthaler J, Glockner FO, Amann R (2000) Cultural ability and in situ abundance of pelagic bacteria from the North Sea. Appl Environ Microbiol 66:3044–3051

<figure><img src="image_21.png" /></figure>

* Eilers H, Pernthaler J, Peplies J, Glockner FO, Gerdts G, Amann R (2001) Isolation of novel pelagic bacteria from the German Bight and their seasonal contributions to surface picoplankton. Appl Environ Microbiol 67: 5134–5142

<figure><img src="image_22.png" /></figure>

* Ewing B, Hillier L, Wendl M, Green P (1998) Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res 8:175–185
* Fandino L, Riemann L, Steward G, Long R, Azam F (2001) Variations in bacterial community structure during a dinoflagellate bloom analyzed by DGGE and 16S rDNA sequencing. Aquat Microb Ecol 23:119–130

* Fuhrman JA, Hewson I, Schwalbach MS, Steele JA, Brown MV, Naeem S (2006) Annually reoccurring bacterial communities are predictable from ocean conditions. Proc Natl Acad Sci USA 103:13104–13109

* Ghiglione JF, Larcher M, Lebaron P (2005) Spatial and temporal scales of variation in bacterioplankton community structure in the NW Mediterranean Sea. Aquat Microb Ecol 40:229–240

* Glöckner FO, Fuchs BM, Amann R (1999) Bacterioplankton compositions of lakes and oceans: a first comparison based on fluorescence in situ hybridization. Appl Environ Microbiol 65:3721–3726

* Granger J, Price N (1999) The importance of siderophores in iron nutrition of heterotrophic marine bacteria. Limnol Oceanogr 44:541–555

* Guieu C, Dulac F, Desboeufs K, Wagener T and others (2010) Large clean mesocosms and simulated dust deposition: a new methodology to investigate responses of marine oligotrophic ecosystems to atmospheric inputs. Biogeosciences 7:2765–2784

* Herut B, Zohary T, Krom M, Mantoura R and others (2005) Response of East Mediterranean surface water to Saharan dust: on-board microcosm experiment and field observations. Deep-Sea Res II 52:3024–3040

* Hewson I, Steele JA, Capone DG, Fuhrman JA (2006) Temporal and spatial scales of variation in bacterioplankton assemblages of oligotrophic surface waters. Mar Ecol Prog Ser 311:67–77

* Hill PG, Zubkov M, Purdie DA (2010) Differential responses of Prochlorococcus and SAR11-dominated bacterioplankton groups to atmospheric dust inputs in the tropical Northeast Atlantic Ocean. FEMS Microbiol Lett 306:82–89

* Hugenholtzt P, Huber T (2003) Chimeric 16S rDNA sequences of diverse origin are accumulating in the public databases. Int J Syst Evol Microbiol 53:289–293

* Ivars-Martinez E, Martin-Cuadrado A, D'Auria G, Mira A and others (2008) Comparative genomics of two ecotypes of the marine planktonic copiotroph Alteromonas macleodii suggests alternative lifestyles associated with different kinds of particulate organic matter. ISME J 2:1194–1212

* Jickells TD, An ZS, Andersen KK, Baker AR and others (2005) Global iron connections between desert dust, ocean geochemistry, and climate. Science 308:67–71

* Jurado E, Dachs J, Duarte C, Simó R (2008) Atmospheric deposition of organic and black carbon to the global oceans. Atmos Environ 42:7931–7939

* Kan J, Evans SE, Chen F, Suzuki MT (2008) Novel estuarine bacterioplankton in rRNA operon libraries from the Chesapeake Bay. Aquat Microb Ecol 51:55–66

Klepac-Ceraj V, Ceraj I, Polz MF (2006) Clusterer: extendable java application for sequence grouping and cluster analyses. Online J Bioinformatics 7:15–21

* Kraemer S (2004) Iron oxide dissolution and solubility in the presence of siderophores. Aquat Sci 66:3–18

* Laghdass M, West NJ, Batailler N, Caparros J and others (2010) Impact of lower salinity waters on bacterial heterotrophic production and community structure in the offshore NW Mediterranean Sea. Environ Microbiol Rep 2:761–769

* Lami R, Ghiglione JF, Desdevises Y, West NJ, Lebaron P (2009) Annual patterns of presence and activity of marine bacteria monitored by 16S rDNA–16S rRNA fingerprints in the coastal NW Mediterranean Sea. Aquat Microb Ecol 54:199–210

* Lamy D, Obernosterer I, Laghdass M, Artigas LF and others (2009) Temporal changes of major bacterial groups and bacterial heterotrophic activity during a *Phaeocystis globosa* bloom in the eastern English Channel. Aquat Microb Ecol 58: 95–107

* Lebaron P, Parthuisot N, Catala P (1998) Comparison of blue nucleic acid dyes for flow cytometric enumeration of bacteria in aquatic systems. Appl Environ Microbiol 64: 1725–1730

* Lebaron P, Servais P, Troussellier M, Courties C and others (2001) Microbial community dynamics in Mediterranean nutrient-enriched seawater mesocosms: changes in abundances, activity and composition. FEMS Microbiol Ecol 34:255–266

* Lee DH, Zo YG, Kim SJ (1996) Nonradioactive method to study genetic profiles of natural bacterial communities by PCR-single-strand-conformation polymorphism. Appl Environ Microbiol 62:3112–3120

* Lekunberri I, Lefort T, Romero E, Vazquez-Dominguez E and others (2010) Effects of a dust deposition event on coastal marine microbial abundance and activity, bacterial community structure and ecosystem function. J Plankton Res 32:381–396

* López-López A, Bartual S, Stal L, Onyshchenko O, Rodríguez-Valera F (2005) Genetic analysis of housekeeping genes reveals a deep-sea ecotype of *Alteromonas macleodii* in the Mediterranean Sea. Environ Microbiol 7:649–659

* Ludwig W, Strunk O, Westram R, Richter L, Meier H (2004) ARB: a software environment for sequence data. Nucleic Acids Res 32:1363–1371

* Maidak BL, Cole JR, Lilburn TG, Parker CT Jr and others (2001) The RDP-II (ribosomal database project). Nucleic Acids Res 29:173–174

Manz W, Amann R, Ludwig W, Wagner M, Schleifer KH (1992) Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions. Syst Appl Microbiol 15:593–600

* Manz W, Amann R, Ludwig W, Vancanneyt M, Schleifer K (1996) Application of a suite of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylum Cytophaga-Flavobacter-Bacteroides in the natural environment. Microbiol 142:1097–1106

Marie D, Partensky F, Simon N, Guillou L, Vaulot D (2000) Flow cytometry analysis of marine picoplankton. In: De Maggio S (ed) Living color: protocols in flow cytometry and cell sorting. Springer Verlag, Berlin, p 421–454

Maring H, Savoie D, Izaguirre M, Custals L, Reid J (2003) Mineral dust aerosol size distribution change during atmospheric transport. J Geophys Res 108, 8592, doi: 8510.1029/2002JD002536

* Mary I, Cummings DG, Biegala IC, Burkill PH, Archer SD, Zubkov MV (2006) Seasonal dynamics of bacterioplankton community structure at a coastal station in the western English Channel. Aquat Microb Ecol 42:119–126

* Mills MM, Ridame C, Davey M, La Roche J, Geider RJ (2004) Iron and phosphorus co-limit nitrogen fixation in the eastern tropical North Atlantic. Nature 429:292–294

* Morris RM, Rappé MS, Connon SA, Vergin KL, Siebold WA, Carlson CA, Giovannoni SJ (2002) SAR 11 clade dominates ocean surface bacterioplankton communities. Nature 420:806–810

* Perenthaler A, Perenthaler J, Amann R (2002) Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria. Appl Environ Microbiol 68:3094–3101

* Pinhassi J, Gómez-Consarnau L, Alonso-Sáez L, Sala MM, Vidal M, Pedrós-Alió C, Gasol JM (2006) Seasonal
changes in bacterioplankton nutrient limitation and their effects on bacterial community composition in the NW Mediterranean Sea. Aquat Microb Ecol 44:241–252

► Pukall R, Päuker O, Buntefuß D, Ulrichs G and others (1999) High sequence diversity of *Alteromonas macleodii*-related cloned and cellular 16S rDNAs from a Mediterranean seawater mesocosm experiment. FEMS Microbiol Ecol 28: 335–344

► Pulido-Villena E, Wagener T, Guieu C (2008) Bacterial response to dust pulses in the western Mediterranean: implications for carbon cycling in the oligotrophic ocean. Global Biogeochem Cycles 22, GB1020, doi:10.1029/2007GB003091

► Pulido-Villena E, Rérolle V, Guieu C (2010) Transient fertilizing effect of dust in P-deficient LNLC surface ocean. Geophys Res Lett 37, L01603, doi:10.1029/2009GL041415
Reche I, Ortega-Retuerta E, Romera O, Pulido-Villena E, Morales-Baquero R, Casamayor E (2009) Effect of Saharan dust inputs on bacterial activity and community composition in Mediterranean lakes and reservoirs. Limnol Oceanogr 54:869–879

► Ridame C, Guieu C (2002) Saharan input of phosphate to the oligotrophic water of the open western Mediterranean Sea. Limnol Oceanogr 47:856–869

► Schäfer H, Servais P, Muyzer G (2000) Successional changes in the genetic diversity of a marine bacterial assemblage during confinement. Arch Microbiol 173:138–145

► Schäfer H, Bernard L, Courties C, Lebaron P and others (2001) Microbial community dynamics in Mediterranean

nutrient-enriched seawater mesocosms: changes in the genetic diversity of bacterial populations. FEMS Microbiol Ecol 34:243–253

► Schloss PD, Larget BR, Handelsman J (2004) Integration of microbial ecology and statistics: a test to compare gene libraries. Appl Environ Microbiol 70:5485–5492

► Seitzinger S, Sanders R (1999) Atmospheric inputs of dissolved organic nitrogen stimulate estuarine bacteria and phytoplankton. Limnol Oceanogr 44:721–730

► Sekar R, Pernthaler A, Pernthaler J, Warnecke F, Posch T, Amann R (2003) An improved protocol for quantification of freshwater Actinobacteria by fluorescence *in situ* hybridization. Appl Environ Microbiol 69:2928–2935

► Shaw AK, Halpern AL, Beeson K, Tran B, Venter JC, Martiny JBH (2008) It's all relative: ranking the diversity of aquatic bacterial communities. Environ Microbiol 10: 2200–2210

► Wagener T, Guieu C, Leblond N (2010) Effects of dust deposition on iron cycle in the surface Mediterranean Sea: results from a mesocosm seeding experiment. BiogeoSCIENCES Discuss 7:2799–2830

► West NJ, Obernosterer I, Zemb O, Lebaron P (2008) Major differences of bacterial diversity and activity inside and outside of a natural iron-fertilized phytoplankton bloom in the Southern Ocean. Environ Microbiol 10:738–756

► Zemb O, Haegeman B, Delgenes J, Lebaron P, Godon J (2007) Safum: statistical analysis of SSCP fingerprints using PCA projections, dendrograms and diversity estimators. Mol Ecol Notes 7:767–770