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1810 Limnol. Oceanogr., 50(6), 2005, 1810–1819 q 2005, by the American Society of Limnology and Oceanography, Inc. Effect of atmospheric nutrients on the autotrophic communities in a low nutrient, low chlorophyll system Sophie Bonnet,1 Cecile Guieu, Jacques Chiaverini, Josephine Ras, and Agnes Stock Laboratoire d'Oceanographie de Villefranche (LOV) La Darse, BP 08, 06238 Villefranche-sur-mer, CEDEX, France Abstract The effect of atmospheric inputs on phytoplanktonic dynamics was investigated in the Mediterranean Sea during the season characterized by a stratified water column, low primary productivity, and low concentrations of nutrients ([nitrate] , 50 nmol L21; [phosphate] 5 20 nmol L21; [silicate] 5 0.7 mmol L21). We report here data obtained during microcosm enrichment experiments performed on the natural assemblage using different combinations of realistic additions (Saharan dust, Fe, Fe 1 phosphate, and anthropogenic particles). Saharan dust and Fe 1 phosphate treatments significantly stimulated primary production. Anthropogenic particles and Fe 1 phosphate treatments increased the chlorophyll a concentrations, enhancing mainly the small cells (pico- and nanophytoplankton). The autotrophic community structure was significantly altered; for example, Fe and Fe 1 phosphate additions benefited prokaryotic populations, indicating possible nitrogen fixation. The colimitation of both phosphate and Fe was re- moved by these additions.
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1810 Limnol. Oceanogr., 50(6), 2005, 1810–1819 q 2005, by the American Society of Limnology and Oceanography, Inc. Effect of atmospheric nutrients on the autotrophic communities in a low nutrient, low chlorophyll system Sophie Bonnet,1 Cecile Guieu, Jacques Chiaverini, Josephine Ras, and Agnes Stock Laboratoire d'Oceanographie de Villefranche (LOV) La Darse, BP 08, 06238 Villefranche-sur-mer, CEDEX, France Abstract The effect of atmospheric inputs on phytoplanktonic dynamics was investigated in the Mediterranean Sea during the season characterized by a stratified water column, low primary productivity, and low concentrations of nutrients ([nitrate] , 50 nmol L21; [phosphate] 5 20 nmol L21; [silicate] 5 0.7 mmol L21). We report here data obtained during microcosm enrichment experiments performed on the natural assemblage using different combinations of realistic additions (Saharan dust, Fe, Fe 1 phosphate, and anthropogenic particles). Saharan dust and Fe 1 phosphate treatments significantly stimulated primary production. Anthropogenic particles and Fe 1 phosphate treatments increased the chlorophyll a concentrations, enhancing mainly the small cells (pico- and nanophytoplankton). The autotrophic community structure was significantly altered; for example, Fe and Fe 1 phosphate additions benefited prokaryotic populations, indicating possible nitrogen fixation. The colimitation of both phosphate and Fe was re- moved by these additions.
- bottles compared
- also below
- atmospheric nutrients
- addition
- dphosphate increased linearly
- after introduction
- such low-nutrient
- french dyfamed
Limnol. Oceanogr.,50(6), 2005, 1810±1819 q2005, by the American Society of Limnology and Oceanography, Inc.
Effect of atmospheric nutrients on the autotrophic low chlorophyll system
communities in a low nutrient,
1 Sophie Bonnet, CeÂcile Guieu, Jacques Chiaverini, JosÂephine Ras, and AgnÁes Stock Laboratoire d'OceÂanographie de Villefranche (LOV) La Darse, BP 08, 06238 Villefranche-sur-mer, CEDEX, France
Abstract The effect of atmospheric inputs on phytoplanktonic dynamics was investigated in the Mediterranean Sea during the season characterized by a strati®ed water column, low primary productivity, and low concentrations of nutrients 212121 ([nitrate],50 nmol L ; [phosphate]520 nmol L ; [silicate]50.7m). We report here data obtainedmol L during microcosm enrichment experiments performed on the natural assemblage using different combinations of realistic additions (Saharan dust, Fe, Fe1phosphate, and anthropogenic particles). Saharan dust and Fe1phosphate treatments signi®cantly stimulated primary production. Anthropogenic particles and Fe1phosphate treatments increased the chlorophyllaconcentrations, enhancing mainly the small cells (pico- and nanophytoplankton). The autotrophic community structure was signi®cantly altered; for example, Fe and Fe1phosphate additions bene®ted prokaryotic populations, indicating possible nitrogen ®xation. The colimitation of both phosphate and Fe was re-moved by these additions. Results emphasized the effect of Fe, although the ambient concentration was close to 1 21 nmol L . The addition of dust bene®ted eukaryotic populations, which indicates that the dust was a possible source of nitrogen. An abiotic dissolution experiment of macronutrients attached to dust con®rmed this hypothesis. The dissolution of Fe attached to the dust (0.23±0.61%) and to the anthropogenic particles (0.86±1.85%) was consistent with previous studies conducted under abiotic conditions. This result suggests that the possible enhancement of the dissolution processes caused by biological activity might have been balanced by Fe consumption by the biota and its adsorption on both mineral and organic particles.
Studying the key interactions between the ocean and the anthropogenic aerosols from industrial and domestic activi-atmosphere is essential to understanding the present function ties from populated areas around the basin and other parts of biogeochemical cycles in the ocean and to predict their of Europe. This situation makes the Mediterranean Sea an evolution. Atmospheric deposition is now recognized as a excellent natural laboratory to study the biogeochemical ef-signi®cant source of external iron (Fe) and other nutrients fect of atmospheric inputs on the water column. In the open for surface waters. Although the effect of Fe on productivity Mediterranean Sea during the season characterized by a has been recognized in high-nutrient, low-chlorophyll strati®ed water column and a low primary productivity, the (HNLC) regions (e.g., Martin et al. 1994), the ecological atmosphere becomes the main external source of nutrients effects of atmospheric Fe and macronutrients in terms of for the mixed layer, the nutrients being depleted after the species response and community structure in oligotrophic bloom. Therefore, this period of the year appears to be the environments are poorly understood. Natural and anthropo- ideal season for testing the fertilizing potential of the at-genic changes in climate and global biogeochemistry can mosphere to surface waters. alter the atmospheric input of aerosols to the ocean. It is The aim of this study was to experimentally determine the important to understand how these modi®cations cause effect of atmospheric deposition on biomass, community changes in planktonic productivity and food web structure structure, and productivity of autotrophic communities dur-because they could result in altered carbon partitioning and ing the strati®ed period. Different combinations of realistic biogenic air±sea gas ¯uxes. additions were conducted on natural assemblages enclosed The Mediterranean Sea is an oligotrophic quasi-enclosed in microcosms to mimic the natural inputs of macro- and basin receiving the highest rate of aeolian material deposi- micronutrients from the atmosphere. These enrichment ex-tion in the world (Guerzoni et al. 1999) in the form of strong periments were carried out to study the (co)limitations en-pulses of mineral dust. In addition, it continuously receives countered in such low-nutrient, low-chlorophyll systems and to determine to what extent atmospheric inputs can relieve them. Another goal was to specify which phytoplanktonic 1 Corresponding author (sbonnet@obs-vlfr.fr).community (pico-, nano-, or microphytoplankton) took the most advantage of these different inputs. Acknowledgments The work reported is part of the doctoral dissertation of S.B. (grant of the French MinisteÁre de l'Education Nationale, de Materials and methods l'Enseignement SupeÂrieur et de la Recherche). It was supported by the European ADIOS Program (project EVK3-CT-2000-00035). We Water collection and incubationÐThis research was car-thank J. C. Marty, responsible for the French DYFAMED time se-ried out on board R/VTeÂthys IIon 1 August 2003 in the ries program, who facilitated this research, and the captain and crew northwestern Mediterranean Sea at the permanent time series of theTeÂthys IIfor their cooperative work at sea. We also thank O. DYFAMED site (438259N, 78529E, 50 km off Nice, France; Bonilla for counting bacteria and G. Sarthou, H. Claustre, and two anonymous reviewers for their helpful comments and suggestions.Fig. 1). This open sea site (2,350 m depth) is protected from We thank K. Forbes for editing the manuscript.coastal inputs by the presence of the coastal Ligurian current. 1810
Fig. 1.
Atmospheric nutrients and phytoplankton
Location of the DYFAMED time series station.
Seawater was collected at 10-m depths (above the thermo-cline) with a trace metal±clean Te¯on pump system and was dispensed into acid-washed 4.5-liter transparent polycarbon-ate microcosms under a laminar ¯ow hood. Filtered seawater (Sartorius Sartrobran-P-capsule 0.45-mm pre®lter and 0.2-mm ®nal ®lter) was also collected to analyze dissolved iron (DFe) concentration in surface seawater before the experi-ment. The bottles were immediately amended with Saharan dust 2121 (0.25 mg L ), anthropogenic particles (0.01 mg L ), Fe 2121 (2.5 nmol L ), and Fe1phosphate (2.5 nmol L Fe, 0.18 21 mphosphate). One unamended treatment was keptmol L as a control. Each fertilization was performed in duplicate. The amount of Saharan dust and anthropogenic particles added was extremely low so that the realistic atmospheric inputs in the Mediterranean Sea could be reproduced. The concentrations of phosphate were similar to those encoun-tered during the winter season in the mixed layer at the DY-FAMED site according to Marty et al. (2002). For Fe, the addition corresponded to the concentration found in the mixed layer after a series of Saharan dust events (Guieu et al. 2002a). The dust we used was composed of ®ne fractions of surface soils collected in the Hoggar region (south Al-geria) and was representative of the Saharan aerosol carried over the western Mediterranean Sea (Guieu et al. 2002b). A standard reference material of the National Institute of Stan-dards and Technology named ``Urban particulate matter'' was used as a proxy for anthropogenic aerosol. The bottles were capped, sealed with polyvinyl chloride tape, and incubated onshore in two outdoor tanks at;50% ambient light level to reproduce the light conditions at the depth at which seawater was collected. A running seawater system continuously supplied water from the sea surface to maintain a constant temperature (258C). For each experi-mental treatment, duplicates were discarded at three selected time points during the course of the experiment (T1520 h; T2544 h; T3568 h) to minimize the risk of contam-ination from handling. Subsamples were used for the follow-ing measurements: chlorophylla(Chla); pigments analysis; primary production; bacterial abundances; DFe concentra-tions, which were only measured in the microcosms amend-ed with Saharan dust; and anthropogenic particles to quan-tify Fe dissolution from these particles.
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Dissolution experimentÐDissolution in seawater of Fe at-tached to the Saharan dust and anthropogenic particles used in this study was parameterized previously in Bonnet and Guieu (2004). To extend this knowledge, the dissolution pro-cesses of nitrate, phosphate, and silicate attached to these particles were also measured. Increasing amounts (eight con-21 centrations between 0.01 and 10 mg L ) of Saharan dust and anthropogenic particles were added to natural ®ltered seawater (0.2mm). After 7 d of incubation, the samples were passed through a 0.2-mm ®lter to determine the dissolved fraction of each nutrient. For details on methods and pro-cedures,seeBonnet and Guieu (2004).
Analytical methodsÐChlaand accessory pigments were measured by high-performance liquid chromatography. Sam-ples (1 liter ®ltered through GF/F ®lters) were extracted in 3 mL of methanol and injected onto a reversed-phase chro-matographic column and analyzed according to the protocol described by Claustre et al. (2004). Carbon assimilation was 14 performed with the C radiocarbon technique (Marty and Chiaverini 2002). Bacterial abundance was determined by epi¯uorescence microscopy as described in Noble and Fuhr-man (1998). Duplicate samples of 0.8 mL were ®ltered onto 25-mm 0.02-mm ®lters (Anodisc, Whatmann) backed by wet 25-mm 0.45-mm pre®lters (Millipore). Twenty randomly se-lected ®elds were counted so that there were at least 200 bacteria per ®lter. Nitrate, phosphate, and silicate in the bulk water used to perform the experiment were analyzed by the automated TechnicontEvolution II (Alliance Instruments) according to standard automated colorimetric methods (TreÂ-guer and Le Corre 1975). Analytical precision for these mea-2121 surements was 20 nmol L (phosphate) and 50 nmol L (nitrate and silicate). DFe concentrations were determined by ¯ow injection analysis with chemiluminescence detection (Obata et al. 1993) on ®ltrate (0.2mm) in each duplicate bottle amended with Saharan and anthropogenic particles. The samples were acidi®ed with HCl Ultra Pure (Merck) at pH 2 and stored in the dark until analyzed. The detection 2121 limit was 20 pmol L and the blank was 80 pmol L . The accuracy of the method was accessed with NASS-5 reference 21 seawater (3.586certi®ed value, 3.910.60 nmol L 60.04 21 nmol L determined value,n53).
Results
Characteristics of the study site at the time of the exper-imentÐDuring the mission, the water column was well strat-i®ed with a marked thermocline at 14 m depth. The surface waters were depleted in nutrients, with phosphate concentra-21 tion close to the detection limit (20 nmol L ), nitrate con-21 centrations of,50 nmol L , and silicic acid concentrations 21 of 0.7mmol L . The total Chla(TChla) concentration (Chla1divinyl Chla), a universal indicator of phytoplank-21 tonic biomass, was 0.05mg L at 10 m depth, and phyto-planktonic biomass was dominated by picophytoplankton (41%) and nanophytoplankton (40%) communities. The con-tribution of microphytoplankton to the total phytoplankton biomass was relatively small (19%). 21 Total DFe concentration was 0.9060.02 nmol L in the surface layer. The reason for this high DFe concentration
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Bonnet et al.
Table 1. Results of the statistical comparison between the control and each treatment at the end of the experiment (Mann±Whitney U-test) for Chlaand primary production. No data for primary pro-duction for addition with anthropogenic particles (samples lost).
Fe Dust FeP Anth ChlaS SNS NS Primary production NS S S Lost NS, not signi®cant (i.e, the difference between the two treatments is not statistically different); S, signi®cant (i.e., the difference between the two treatments is statistically different); FeP, Fe1phosphate; Anth, anthro-pogenic particles.
21 (compared with,0.13 nmol L for the same period in May 1995; Sarthou and Jeandel 2001) could not be explained by the small inputs from Saharan events that had occurred since the beginning of strati®cation: one Saharan event of low am-22 plitude (0.288 g m ) had been recorded before the experi-ment at the Cap Ferrat atmospheric station (6 km from Nice, France), and three small events were recorded in Corsica, 22 France, accounting for a total dust ¯ux of 0.310 g m (M.D. LoyÈ e-Pilot pers. comm.). Rather, this DFe concentration was attributed to the forest ®res that occurred in the south of France continuously for 1 month before the experiment (Guieu et al. unpubl.).
Biological response during incubationÐFor each param-eter measured, the statistical Mann±Whitney U-test (5% er-ror rate) was used to determine whether concentrations in the bottles amended with nutrients were signi®cantly differ-ent from those in the control bottles (Tables 1, 3).
Chla: Additions of Fe1phosphate and anthropogenic particles had a signi®cant effect on phytoplankton biomass over the duration of the experiment (Table 1; Fig. 2). It did not change signi®cantly in the treatments amended with dust or Fe.
Primary production: Primary production was stimulated in most of the enriched bottles compared with the control over the incubation time (Fig. 3). The responses were sig-ni®cantly higher in the treatments amended with dust and Fe1phosphate than in the control: primary production in-tegrated over the experimental period increased by 48% and 56%, respectively, compared with the unamended treatment. Production also increased after Fe addition, but in a lower proportion (123%) that was not found to be statistically dif-ferent from the unamended treatment. Primary production after the addition of anthropogenic particles could not be measured (because of human error, the samples were lost).
Phytoplankton community: Evolution of the phytoplank-ton community, divided into three representative size classes (Table 2) after the additions, was examined by pigment anal-ysis (Fig. 4; Table 3). The biomass proportion of TChla associated with picophytoplankton (,2mm, BPpico), nan-ophytoplankton (2±20mm, BPnano), and microphytoplank-ton (20±200mm, BPmicro) was calculated as described by Vidussi et al. (2001) and revisited by Uitz et al. (unpubl.): BPpico5[0.86 (zea)11.01 (TChlb)]/DP, where DP is the
Fig. 2. Concentrations of Chlain unamended and nutrient-amended treatments in the course of incubation. The error bars rep-resent the standard deviation from duplicate incubations.
sum of diagnostic pigments [DP50.86 (zea)11.01 (TChl b)10.60 (allo)11.27 (199HF)10.35 (199BF)11.41 (fuco)11.41 (peri)], BPnano5[0.60 (allo)11.27 (199HF) 10.35 (199BF)]/DP, and BPmicro5[1.41 (fuco)11.41 (peri)]/DP (see Table 2for variable de®nitions). It should be noted that the range of variation of each pig-ment concentration was very narrow. The results obtained must therefore be considered with caution, especially for mi-crophytoplankton, because the concentrations of fucoxanthin and peridinin measured were close to the average detection limit of the method. Picophytoplankton revealed the most pronounced re-sponse to Fe additions between T0 and T3 (Fig. 4a); pico-phytoplankton dominated the entire community at the end of the experiment (Table 3); its proportion was signi®cantly higher than in the control bottle at T3. Nanophytoplankton was stimulated in the treatments amended with Fe1phos-phate and anthropogenic particles; their proportions were also signi®cantly higher than in the unamended treatment at T3 (Table 3). The biomass proportion associated with mi-
Fig. 3. Primary production integrated over the incubation pe-riod in nutrient-amended treatments and normalized to the control. The error bars represent the standard deviation from duplicate in-cubations.
Atmospheric nutrients and phytoplankton
Table 2. Biomarker pigments used in the present study, their abbreviations, taxonomic signi®cance, and associated size class.
Diagnostic pigment Fucoxanthin Peridinin 199Hexanoyloxyfucoxanthin 199Butanoyloxyfucoxanthin Alloxanthin Chlb1divinyl-Chlb Zeaxanthin
Abbreviation fuco peri 199HF 199BF allo TChlb zea
Taxonomic signi®cance Diatoms Dino¯agellates Chromophytes nano¯agellates Chromophytes nano¯agellates Cryptophytes Green ¯agellates and prochlorophytes Cyanobacteria and prochlorophytes
crophytoplankton at T3 was not statistically different in the different treatments compared with the control.
Bacterial abundances: Bacterial abundances increased be-tween T1 and T2 and decreased after T2 in all treatments and in the control bottle. Addition of anthropogenic particles to surface seawater had a slight positive effect on the bac-terial abundances (110% relative to the control between T1 and T2). The other treatments did not change bacterial abun-dances.
Potential release of Fe, nitrate, phosphate, and silicate from Saharan dust and anthropogenic particlesÐIn this sec-tion, we examine the potential release of macronutrients ni-trate, phosphate, and silicate and micronutrient Fe from the two types of particles that were used to simulate atmospheric input. For Fe, the numbers presented here represent the ac-tual concentrations measured in the microcosm along the duration of the experiment (Table 4). These numbers will be compared in a future section to those obtained in abiotic conditions by Bonnet and Guieu (2004). For nitrate, phos-phate, and silicate, we show in the following section the results obtained from the dissolution in abiotic conditions (Fig. 5a,b).
Iron: The values of DFe increased in the treatments amended with Saharan dust and anthropogenic particles (Ta-ble 4). Values ofDDFe ([DFe]after introduction of particles2[DFe]initial) were used to calculate the percentage of Fe released by the particles. Considering an Fe content of 5% in the Saharan dust used in these experiments (Guieu et al. 2002b) and 3.91% in anthropogenic particles, the dissolution rates of Fe over the duration of the experiment were between 0.23% and 0.61% for the Saharan dust and between 0.86% and 1.85% for the anthropogenic particles (Table 4).
Nitrate: In our dissolution experiments conducted in abiotic conditions, the values ofDnitrate ([nitrate]aft er introduction of particles 2[nitrate]initial) increased with increasing amounts of parti-cles introduced and followed a linear relationship after the addition of Saharan dust (Fig. 5a) and anthropogenic parti-2 cles (Fig. 5b) (r50.90 and 0.96, respectively).DNitrate 21 ranged from 0.5 to 1.2mmol L after Saharan dust addition 21 and 0.15 to 5.4mafter urban particle addition. Themol L 21 introduction of 0.25 mg L dust resulted in an increase in 21 nitrate of 0.54min our microcosm, and the intro-mol L 21 duction of 0.01 mg L anthropogenic particles resulted in 21 an increase of 0.24m.mol L
Size range (mm)
.20
2±20
,2
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Phytoplankton size class
Microphytoplankton
Nanophytoplankton
Picophytoplankton
Phosphate: From the same experiment,Dphosphate after introduction of particles2initialelow ([phosphate] [phosphate] ) was b 21 the detection limit (,) after the introduction of20 nmol L Saharan dust. After addition of anthropogenic particles, Dphosphate was also below the detection limit for particle 21 concentrations,.1 mg L DPhosphate increased linearly 21 2 for particle concentrations.(1 mg L r50.99) ranging 21 from 0.13 to 1.98m(Fig. 5b).mol L
Silicate:DSilicate ([silicate]after introduction of particles2[sili-21 cate]initial) was below the detection limit (,50 nmol L ) after the introduction of Saharan dust. After addition of anthro-pogenic particles,Dsilicate was also below the detection lim-21 it for particle concentrations below 3 mg L and increased 21 2 linearly for particle concentrations above 3 mg L (r5 21 0.99) ranging from 0.22 to 0.65m(Fig. 5b).mol L
Discussion
Intensity of biological responseÐAccording to the pro-ductivity of the whole community, the magnitude of the bi-ological response obtained after these atmospheric-like ad-ditions was signi®cant in some of the treatments (1dust, 1Fe,1phosphate). The mean Chlaconcentration in these 21 bottles remained low (;0.065m) and characteristic ofg L the oligotrophic conditions encountered in the northwestern Mediterranean Sea during the summer season (Marty et al. 2002). To improve the understanding of this ecological ef-fect, it is critical to relate this to the intensity of the fertil-ization conducted. Indeed, the key point of this experiment stems from the very low ®nal particle or nutrient concentra-tions added to the natural assemblages to simulate those en-countered in the natural environment under realistic condi-21 tions: the addition of 0.25 mg L of Saharan dust in a 14-m mixed layer is equivalent to a medium-amplitude Saharan event (Ridame and Guieu 2002). Our scienti®c approach was signi®cantly different from the mesoscale Fe enrichments performed in HNLC waters, the aim of which was to prove the limiting character of Fe to primary production. In our experiment, we tried to mimic the natural deposition of mineral substances and anthropo-genic particles to examine the capacity of a system, poten-tially limited by several nutrients, to react to a realistic at-mospheric fertilization. To better understand the ecological response of the ecosystem, it is critical to examine which autotrophic community took the most advantage of the ad-ditions.
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Bonnet et al.
Fig. 4. Increase in Chlaconcentrations with incubation time in the three different phytoplankton size classesÐ(a) picophytoplank-ton, (b) nanophytoplankton, and (c) microphytoplanktonÐderived from pigment analysis. The error bars represent the standard devi-ation from duplicate incubations. Note the different scales on they-axis.
Effect on community structureÐThe absence of any major effect of the additions on bacterial abundance obtained in this experiment is probably due to the regulation of the bac-terial biomass development by the heterotrophic nano¯agel-lates (Thingstad et al. 1998). Ridame (2001) also found that dust addition had no major effect on the bacterial abundance.
Table 3. Biomass proportion (BP) of TChlaassociated with each size class of phytoplankton at the end of the incubation period (T3) in the control bottles and the nutrient-amended treatments.
BP (%) Picophyto- Nanophyto- Microphyto-plankton plankton plankton Control 39±43 26±28 29±35 Fe 50±62* 14±25 24±25 Dust 42±43 25±30 27±33 FeP 40±48 28±34* 24±26 Anth 35±37 32±39 26±30 Anth, anthropogenic particles; FeP, Fe1phosphate. * Values were statistically different from the control at T3.
However, she showed that bacterial production was clearly stimulated and proportional to the amount of dust introduced 21 (increase of 70% relative to the control for a 10 mg L addition after 48 h). To understand how the additions affected the different communities, we estimated the part of TChlaassociated with prokaryotic and eukaryotic populations. The prokary-otic proportion of TChlawas calculated as the sum of di-vinyl Chlafrom prochlorophytes and ChlafromSynecho-coccus(calculated with Chla: zeaxanthin51.65 (from Morel et al. 1993). It should be noted that we did not detect divinyl Chlain our sample. Indeed, prochlorophytes are speci®cally abundant at depth at the end of the strati®cation period, usually in late fall (Marty et al. 2002). The eukary-otic proportion of TChlawas obtained by taking the dif-ference of TChlaand prokaryotic Chla.Despite the re-maining ``oligotrophic conditions,'' we observed a shift of the communities in the amended bottles: at T3, the Chla associated with eukar yotes increased in the treatment amended with dust (112%), whereas prokaryotes increased in the bottles amended with Fe and Fe1phosphate (127% and116%, respectively). Because of the absence of prochlo-rophytes, the increase of prokaryotes is necessarily associ-ated with cyanobacteria.
Nutrient supply and requirementsÐDuring the strati®ed period in the Mediterranean Sea, phosphate bioavailability appears to be the main limiting macronutrient for phyto-plankton (Thingstad and Rassoulzadegan 1995) and bacterial production (Van Wambeke et al. 2002). However, the ex-21 tremely low concentrations of nitrate (,50 nmol L ) in the surface waters before this experiment could suggest the ex-istence of nitrate and phosphate colimitations and explain the low intensity of the responses observed. The role of iron could also be important because it has been known to play an important role in the nitrogen ®xation process (see,e.g., Falkowski 1997). Although DFe concentration in the surface 21 mixed layer was ``high'' (0.9 nmol L ) at the time of our experiment, this does not mean that a high fraction of this Fe was bioavailable. As pointed out previously (Blain et al. 2004; Mills et al. 2004) for studies conducted in an envi-ronment subjected to deposition of dust (which can have high dissolved Fe concentrations), the total dissolved con-centration is a poor index of bioavailability. As emphasized
Atmospheric nutrients and phytoplankton
Table 4. Dfe concentrations and percent dissolved Fe (%Dis) associated with Saharan dust and anthropogenic particles for each replicate at each time point relative to the control before the ex-periment.
21 [DFe] (nmol L ) TO: 0.90 21 nmol L Dust Anth. T1 1.3760.062%Dis50.2360.06 1.0660.012%Dis51.8560.01 1.5260.012%Dis50.3060.01 1.0260.022%Dis51.3560.02 T2 2.1360.072%Dis50.6160.07 0.9860.032%Dis50,8660.03 1.5760.082%Dis50.3360.08 2.262%Dis516.73* T3 1.2660.052%Dis50.1860.01 1.2660.032%Dis54.3160.03* 1.9860.122%Dis50.5060.10 1.0560.162%Dis51.7260.16 * Contamination.
21 by Wu et al. (2001), concentrations as high as 1 nmol L can present a small bioavailable fraction and can thus be somehow limiting. The interpretation of the results of these experiments in a low-nutrient, low-chlorophyll system provided new insight into the nutrient supply, indirectly (by nitrogen ®xation) and directly (from the particles introduced).
Nitrogen source: The addition of Fe and Fe1phosphate induced an increase of primary productivity (127% and 156% over the 3-d incubation). This ability to grow without
Fig. 5. Dissolution of nutrients from (a) Saharan dust and (b) anthropogenic particles in seawater from in vitro experiments.
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the addition of nitrogen could be explained by two mecha-nisms acting singly or together: ef®cient utilization of (un-detectable) dissolved inorganic nitrogen or effective nitrogen ®xation. The Chlaincreases measured after Fe and Fe1phos-21 phate additions were 0.027 and 0.013mg L , respectively. 21 With a C : Chlaratio of 6.4 mol C (g Chlaa N : C) and ratio of 16 : 106, the predicted uptake of nitrate would be 13 21 and 26 nmol L , which is undetectable by our analyzer (de-21 tection limit5). The utilization of nitrate, am-50 nmol L monium, or both is thus a possible explanation for the ob-served Chlaincrease in these treatments. It is supposed that such a system is based on regeneration processes during the strati®cation period, but the only data on nitrogen regeneration available in the Mediterranean Sea were established in a coastal zone (Gulf of Lion): a high 2121 ammonia regeneration rate (up to 220 nmol L d ) was found to be suf®cient to sustain the ammonia plankton de-mand (Diaz and Raimbault 2000). Even if such a regenera-tion process is suspected to be lower in the open site where the experiment was performed, regenerated sources of nitro-gen cannot thus be excluded. Nitrogen ®xation also has to be considered a possible sourceofnitrogen.BeÂthouxandCopin-MontÂegut(1986) made the hypothesis of strong nitrogen ®xation processes to explain the net balance of nitrogen at the scale of the Med-iterranean basin. Recent work with measurements of natural isotopic ratios con®rmed this hypothesis in the oriental basin (Sachs and Repeta 1999) and in the occidental basin (Ker-herve et al. 2001). The ®lamentous cyanobacteriumTricho-desmiumis assumed to be the predominant oceanic N2-®xing microorganism (Capone et al. 1997), but it is not abundant in the Mediterranean Sea (BeÂthoux and Copin-Montegut 1986). The role of unicellular cyanobacteria in N2®xation is taken into less consideration, although it is now recog-nized that these microorganisms express nitrogenase (Zehr et al. 2001). These N2-®xing unicellular cyanobacteria are widely distributed in marine environments at concentrations 21 of up to 1,000 cells mL (Neveux et al. 1999) and poten-tially have a signi®cant role in nitrogen dynamics in the world ocean. As we have shown before, picophytoplanktonic popula-tions grew signi®cantly in the Fe treatment and pro®ted the most from this Fe addition. The increase of cyanobacteria proportion in this treatment (127% compared with the con-
