Response of a phytoplankton community to increased nutrient inputs: A mesocosm experiment in the Bay...

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Response of a phytoplankton community to increased nutrient inputs:A mesocosm experiment in the Bay of Brest (France)

Pierre Fouillaron a,⁎, Pascal Claquin b, Stéphane L'Helguen c, Pierre Huonnic a,Véronique Martin-Jézéquel d, Annick Masson a, Sorcha Ní Longphuirt a,

Philippe Pondaven a, Gérard Thouzeau a, Aude Leynaert a

a Laboratoire des Sciences de l'Environnement Marin, UMR 6539 CNRS, IUEM, Université de Bretagne Occidentale,Technopôle Brest-Iroise, 29280 Plouzané, France

b Laboratoire de Biologie et Biotechnologies Marines, UMR 100 IFREMER, Université de Caen Basse-Normandie, 14032 Caen cedex, Francec Laboratoire de Chimie Marine, UMR 7144 CNRS/UPMC, Station Biologique de Roscoff, BP 74, 29682 Roscoff cedex, France

d Laboratoire de Biologie Marine, EA2663, Université de Nantes, BP 92208, 44322 Nantes, France

Received 8 September 2006; received in revised form 15 June 2007; accepted 20 June 2007

Abstract

In the Bay of Brest (France), benthic fluxes of silicic acid (DSi) are thought to be strongly involved in diatom dominance andtheir intensity depends on the density of an invasive gastropod, Crepidula fornicata. While the proliferation of this gastropod islikely to support diatom dominance, local fishermen have proposed its removal as it perturbs the development of the native GreatScallop, Pecten maximus. However, such a removal may be associated with potentially harmful algal blooms. The response of thephytoplankton community to modifications of the benthic fluxes of DSi, resulting from a further proliferation or the removal of C.fornicata, was examined in a mesocosm nutrient enrichment experiment over a period of 16 days in the spring of 2004. Sixmesocosms (5.4 m3) were filled using a natural planktonic assemblage from the Bay and received N, P and Si additions at thestoichiometric ratio of the in situ benthic fluxes (Si:N:P=16:16:1), with a gradient of enrichment ranging from 0 to almost 4 timesthe maximal benthic flux measured in the Bay. The diatom Chaetoceros (mainly C. debilis) rapidly outgrew non-siliceous speciesof microphytoplankton in all enclosures although kinetic experiments provided direct evidence of Si limitation. This dominance ofdiatoms at DSi concentrations b2 μM highlighted that the residual DSi concentration measured daily in the mesocosms is not areliable criterion to predict diatom dominance in marine ecosystems whereas the dependence of the Si uptake rate on fertilizationrates suggests that emphasis should be placed on daily nutrient addition. Quantitative modifications of nutrient additions onlychanged the magnitude of the diatom bloom and high fertilization rates may lead to high sedimentation fluxes of organic material,which can strongly harm the entire benthic community. Finally the diatom dominance at low fertilization rates raised questionsabout the role of benthic fluxes in preventing harmful algal blooms within the Bay system.© 2007 Elsevier B.V. All rights reserved.

Keywords: Benthic fluxes; Diatom dominance; Mesocosm experiment; Silicic acid availability

1. Introduction

The Bay of Brest (France) is a coastal ecosystemwhere Si:N ratios in river inputs have decreased by a

Journal of Experimental Marine Biology and Ecology 351 (2007) 188–198www.elsevier.com/locate/jembe

⁎ Corresponding author. LEMAR — IUEM, Technopôle Brest-Iroise, Place Nicolas Copernic, F – 29280 Plouzané, France. Tel.: +332 98 49 88 99; fax: +33 2 98 49 86 45.

E-mail address: [email protected] (P. Fouillaron).

0022-0981/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.jembe.2007.06.009


factor of three during the past 30 years, mostly becauseof excessive anthropogenic N-inputs (Le Pape et al.,1996). Contrary to a wide variety of coastal ecosystemswhere similar decreases led to a change from diatoms tonon-siliceous species (Smayda, 1990), diatoms stilldominate microphytoplankton until mid summer in theBay (Del Amo et al., 1997).

Benthic fluxes of silicic acid (DSi) are thought to bestrongly involved in this diatom dominance (Ragueneauet al., 2002, 2005). The sedimentation of diatom cellsafter the spring bloom allows Si to be retained within theBay at the sediment–water interface instead of beingexported to the coastal ocean. It then becomes directlyavailable for regenerated diatom production because ofthe shallow depth of the well-mixed waters. Chauvaudet al. (2000) suggested that the functioning of thiscoastal silicate pump was under the control of benthicsuspension feeders and particularly the invasive gastro-pod Crepidula fornicata, which is now the main benthicsuspension feeder in the Bay (Guérin, 2004). The highfiltration activity of C. fornicata leads foremost to theproduction of an enormous quantity of bio-deposits andthen to an increase in the retention of biogenic silica(BSi) in the sediment. Subsequent BSi dissolution isthought to increase the benthic DSi fluxes. This as-sumption was recently validated when benthic fluxes ofSi measured at a site with a high density of C. fornicatawere found to be one order of magnitude higher

than those measured at a site with no C. fornicata(Ragueneau et al., 2002). In spring, the DSi benthicfluxes measured in situ on two sites presenting a lowdensity (b200 individuals m−2) and a high density(N1000 individuals m−2) of C. fornicata were ca 100and 380 μmol m−2 h−1 respectively (Martin, 2005)with a Si:N:P ratio close to the Redfield ratio (Si:N:P=16:16:1). Considering the mean depth (8 m) in theBay of Brest, these benthic fluxes correspond to anincrease of the DSi concentration in the water columnof ca 0.3 and 1.2 μM Si d−1 respectively. In the Bayof Brest, fishermen have proposed the removal ofC. fornicata as it perturbs the development of the nativeGreat Scallop, Pecten maximus (Thouzeau et al., 2000).However the removal of C. fornicata, resulting in alower DSi benthic flux, could also be associated with apotential risk of harmful algal blooms during summermonths (Ragueneau et al., 2002).

In order to test how the modification of the DSibenthic flux intensity may affect the composition of thephytoplankton communities, an enrichment mesocosmexperiment was conducted in spring 2004 using anatural planktonic assemblage obtained from the Bay ofBrest. Six enclosures were filled with seawater andenriched with N, P and Si at the stoichiometric ratioof the in situ benthic fluxes (Si:N:P=16:16:1) with agradient of fertilization. To address the effect of a furtherproliferation of C. fornicata or that of C. fornicata

Fig. 1. Study area and location of the mesocosm mooring site in the Bay of Brest.

189P. Fouillaron et al. / Journal of Experimental Marine Biology and Ecology 351 (2007) 188–198


removal, the gradient of fertilization ranged from 0 toalmost 4 times the maximal benthic flux measured inthe Bay and the in situ Si:N:P ratio was used.

2. Materials and methods

2.1. Study site and mesocosm experiment

The studywas conducted in the Bay of Brest (Fig. 1), asemi-enclosed basin of 180 km2 fed by two tributaries,the Aulne and the Elorn, and connected with the At-lantic Ocean through a narrow (1.8 km wide) and deep(50 m depth) strait. The mean depth is 8 m and more than50% of the total area is b5 m deep. Experiments wereconducted during 16 days, between 24March and 8 April2004.We used 4m deep polyethylene bags (the last meterfrom the bottom of the bags was conical shaped) with avolume of 5.4 m3. Six enclosures (noted E1 to E6) werefilled with seawater from the Bay of Brest by loweringthem into the water column and lifting them up slowly.The time required to fill the six enclosures did notexceed 1 h. Nutrients were added in the evening, from 23March (day 0) until 7 April 2004 (day 15), except inthe unfertilized control, called E1. Fertilizations rateswere defined considering benthic fluxes measured in situ(Martin, 2005) for different densities of C. fornicata.A gradient of addition was performed from enclosureE2 to enclosure E6 using a constant atomic ratio (Si:N:P=16:16:1) (Table 1). Si was in the form of DSi, N asnitrate and P as phosphate. Nutrients were added bylowering a 4 m long flexible tube into the enclosure,filling the tube with the nutrient mixture, which had beenpreviously diluted in seawater to equal the volume of thetube, and finally releasing the nutrient mixture by liftingthe tube. Every 2 days, the bottom-water of the enclosureswas pumped to withdraw sedimented particles.

Water samples were collected at 8:00 a.m. every dayusing a 4 m long tube sampler that allowed sampling ofthe whole water column. Daily measurements of salinity

and temperature were undertaken and checked againstthe outside water profile to ensure that stratificationwas not occurring in the enclosures. 10 L of seawaterwere sampled every day in each enclosure for nutrients,BSi, chlorophyll a, particulate organic nitrogen (PON)concentrations and phytoplankton cell counts and iden-tification. At the end of the experiment, the total amountof water removed from the enclosures was calculated tobe less than 5% of the initial volume of the bags.

2.2. Analytical methods

Nitrate and DSi concentrations were measured im-mediately after sampling on a Technicon Auto AnalyserII (Tréguer and Le Corre, 1975). For phosphate deter-mination, 100 mL of seawater was immediately frozenuntil further analysis by the colorimetricmethod (Murphy

Table 1Daily additions of Si, N and P

Enclosure E1 E2 E3 E4 E5 E6

Si added (μM d−1) 0 0.1 0.5 1.0 2.0 4.0N added (μM d−1) 0 0.1 0.5 1.0 2.0 4.0P added (μM d−1) 0 0.006 0.031 0.063 0.125 0.250

Additions and the nutrient ratio were defined according to benthicnutrient fluxes measured in situ in the Bay of Brest. The nutrient ratiowas kept constant while additions varied through enclosures in order totest the influence of increasing nutrient fluxes. In enclosures E1 to E4,a gradient of addition was determined ranging from zero to themaximal flux measured in the Bay. In enclosures E5 and E6, additionswere 2 and 4 times the maximal flux.

Fig. 2. Evolution of (a) silicic acid (DSi) and (b) biogenic silica (BSi)concentrations in the six enclosures during the experiment conductedin March–April 2004. (c) Synthetic graph of the Si:N:P ratio in thewater column of the six mesocosms. In each area, delimited by theBrzezinski (1985) ratio and the Redfield ratio (Si:N:P=16:16:1), thepotential limiting nutrients are reported in order of priority.

190 P. Fouillaron et al. / Journal of Experimental Marine Biology and Ecology 351 (2007) 188–198


and Riley, 1962). BSi was determined from particu-late matter collected by filtration of 1 L of seawaterthrough 0.6 μm polycarbonate membrane filter. Analysiswas performed using the alkaline digestion method(Ragueneau and Tréguer, 1994). PON concentrationswere measured every 2 days on two size fractions(unfractionated and b10 μm). Samples were gravity-fil-tered through 10 μm Nuclepore filters to get the b10 μmfraction. Particulate material for PON measurementswas recovered on pre-combusted (450 °C, 4 h) WhatmanGF/F filters and analyzed in a Thermoelectron elementalanalyser (Flash EA) using atropine as the standard. PONfor the N10 μm fraction was obtained by subtractionof the b10 μm concentration from the unfractionatedsample. The samples for the determination of chlorophylla concentrations were unfortunately defrosted beforeanalysis could occur. Phytoplankton samples were col-lected every 2 days, fixed with lugol and cell counts werecarried out using the Utermöhl (1958) method. Zoo-plankton was sampled at the beginning and the end of theexperiment by filtering water on an 80 μm filter. At thebeginning of the experiment, only a low volume (60 L)was filtered in order to avoid a drastic removal ofthe initial zooplankton community. At the end of theexperiment, a larger volume (1500 L) was filtered. Sam-ples were then conserved in a solution of 150 mL offiltered seawater containing 2.5% formol and 4 g L−1 ofdi-sodium tetraborate (Na2B4O7·10H2O). It should benoted that at the end of the experiment, the volumefiltered was normalised to 60 L for comparison with theinitial conditions.

The concentration dependence of the diatom silicicacid uptake rate was investigated just after DSi depletion

(DSi≤1 μM) occurred, in enclosure E2 (day 7) andin enclosures E4 and E5 (day 11). For each kineticexperiment, a set of seven 250 mL sub-samples werepoured into incubation bottles and enriched with Si(OH)4 ranging from 0 to 12 μM above ambient con-centrations. Then 40000 dpm (660 Bq) of 32Si tracer(52000 Bq/μg Si, Los Alamos National Laboratory)was added to each bottle. Incubations were undertakenfor 4 h, around solar noon, under simulated in situconditions by using nickel screens that reduced ambientlight transmission by 50%. After incubation, sampleswere filtered onto 0.6 μm Nuclepore filters. Each filterwas then immediately placed in a 20 mL plastic vial. Atthe laboratory, 2 mL of 2.9 M fluoridric acid (HF) wasadded to the vials to dissolve the biogenic silica. After1 h, 15 mL of scintillation liquid (Ultima Gold XR)was added and the radioactivity was counted on a 1414Winspectral scintillation counter (Perkin Elmer) for60 min, or when a counting precision of 0.5% wasachieved for cpm in each counting window. An equil-ibrated Si solution and P standards were used to de-convolve the energy spectra (Leynaert et al., 1996;Brzezinski and Phillips, 1997).

3. Results

3.1. Initial conditions

Initial conditions were measured in all enclosures onday 1. Nitrate, DSi and phosphate concentrations were19.9±0.6 μM, 5.0±0.2 μM and 0.40±0.08 μM,respectively. BSi and total PON concentrations were1.9 ± 0.4 μmol L− 1 and 2.51 ± 0.08 μmol L− 1,

Fig. 3. Evolution of the two-size-fractioned particulate organic nitrogen (PON) concentrations in the six enclosures during the experiment conductedin March–April 2004.



respectively. The cell concentration of microphyto-plankton averaged 5.5±2.2 104 cells L−1. The cellconcentration of nanophytoplankton was two orders ofmagnitude higher and averaged 1.5±0.3 106 cells L−1.The diatom Chaetoceros accounted for 30±14% ofmicrophytoplankton cell counts. The mean abundanceof copepods in the 60 L samples was 101±41 indi-viduals. Copepods (mainly Acartia sp., Pseudocalanusminutus and Temora longicornis) represented 37±9% ofthe total zooplankton counts; the remaining being mostlycomposed of nauplii of copepods and cyripeds.

3.2. Evolution of nutrient concentrations

In E1, E2 and E3, DSi concentrations decreasedslowly until DSi depletion on day 7 (Fig. 2a). In E4, DSiconcentrations did not change until day 5 and thendecreased rapidly until depletion on day 7. In E5 and E6,DSi concentrations increased considerably until day 6with maximum values of 12.7 and 24.5 μM respectively.This increase was followed by a rapid decrease and bothenclosures exhibited DSi depletion after 11 days. DSidepletion continued in all enclosures from day 11 to theend of the experiment. During the experiment, nitrateand phosphate concentrations remained above 3.0 and0.08 μM, respectively. The measured values of Si:N:Pratios suggested that Si was potentially limiting in allenclosures during the entire experiment (Fig. 2c).

3.3. Evolution of BSi and PON concentrations

After 5 days, BSi concentrations increased exponen-tially (Fig. 2b). On day 7, BSi concentrations in E1, E2,E3 and E4 reached maximal values of 3.5, 4.0, 6.7 and

9.0 μmol L−1, respectively. From day 7 to the end ofthe experiment, BSi concentrations in these enclosuresremained almost constant with a small decrease at theend of the experiment. In E5 and E6, maximal BSiconcentrations were reached on day 11 and 12 withvalues of 16.8 and 29.8 μmol L−1, respectively. Sub-sequently, BSi concentrations decreased slowly in E5while a strong decrease occurred in E6. BSi concen-trations at the end of the experiment were 10.4 and13.2 μmol L−1 in E5 and E6 respectively.

The evolution of the two size fractions of PON ispresented in Fig. 3. The PON fraction N10 μm whichrepresented almost 25% of the total at the beginning ofthe experiment, increased rapidly to reach more than80% of the total at the end of the experiment in allenclosures. This PON fraction showed a trend similar tothat of the BSi and a significant correlation ( pb0.01)was measured between these two parameters (Fig. 4).

Fig. 5. Evolution of (a) microphytoplankton and (b) nanophytoplank-ton cell counts and (c) Chaetoceros dominance given as the meanpercentage of microphytoplankton cells counts in the six enclosuresduring the experiment conducted in March–April 2004.

Fig. 4. Biogenic silica (BSi) concentrations as a function of theN10 μm particulate organic nitrogen (PON) concentrations. This figureincludes data collected from the six enclosures. The calculated slope is0.85 (R2=0.94).



Inversely, the b10 μm PON fraction generally decreasedwith time.

3.4. Evolution of phytoplankton abundance andcomposition

Enclosures E1 to E4 were characterized by an expo-nential increase of the microphytoplankton cell number(encompassing diatoms, dinoflagellates and silicofla-gellates) until day 7 followed by a stabilization until theend of the experiment with final cells concentrations of1.0, 1.2, 1.7 and 2.4 106 cells L−1 respectively (Fig. 5a).In E5 and E6, a huge increase occurred between day 7and day 11 with maximal values of 6.0 and 7.8 106 cellsL−1, respectively. This increase is followed by a largedecrease and final concentrations (2.7 and 4.6 106 cells

L−1 respectively) were in the same order of magnitudeas in the other enclosures. In enclosures E1 to E4,nanophytoplankton cell concentrations (encompassingnanoflagellates like Cryptophycaea and small Dinophy-caea) did not change significantly between enclosuresand remained almost constant until the end of the ex-periment with final cells concentrations of 1.6, 1.2, 1.8and 2.1 106 cells L−1, respectively (Fig. 5b). As formicrophytoplankton, a huge increase occurred in E5 andE6 between day 7 and day 11 with maximal values of3.5 and 6.9 106 cells L−1 respectively. This increase isfollowed by a large decrease and final concentrations(2.1 and 1.1 106 cells L−1 respectively) were of the sameorder of magnitude as those in the other enclosures.

In all enclosures, diatoms dominated microphyto-plankton throughout the experiment and after 5 days the

Fig. 6. The (a) copepods (adults+copepodites I–III) and (b) Oikopleura cf. dioica abundances (ind. 60 L−1) at the beginning (white) and at the end(black) of the experiment in the six enclosures during the experiment conducted in March–April 2004. It should be note that at the end of theexperiment, the volume filtered was 1500 L and normalised to 60 L for comparison with the initial conditions.



major genus was Chaetoceros, dominated by the speciesC. debilis. Some Rhizosolenia spp. (mainlyR. imbricata)were present throughout the experiment in all enclo-sures but were rapidly outnumbered by C. debilis thataccounted for 70±9% of the microphytoplankton cellnumbers on day 5 (Fig. 5c). The mean cell volume ofC. debilis was 481±151 μm3.

3.5. Evolution of zooplankton abundance andcomposition

Enclosures E1 to E6 exhibited various initial cope-pod abundances with values ranging from 30 to 139individuals in the 60 L filtered (Fig. 6a), with an over-all average of 101±41 ind. 60 L–1, i.e. 37±9% of thetotal zooplankton counts. At the end of the experiment,the average copepod abundance (100±28 ind. 60 L–1)was not significantly different from the initial values

(Student t-test, p=0.98, n=6) and values ranged from65 to 130 ind. in the 60 L filtered. Acartia sp., P. minutusand T. longicornis remained the dominant speciesthroughout the experiment, reaching 79–91% of thetotal copepod density at the end of the experiment. Someappendicularians, Oikopleura cf. dioica were also ob-served at the beginning of the experiment with a similarlow abundance (b12 ind. 60 L–1) in all enclosures(Fig. 6b). Contrary to copepods, they developed duringthe experiment and a clear density gradient, increasingfrom E1 to E6 was found.

3.6. Kinetic studies of silicic acid uptake

For the three kinetic experiments carried out in E2,E4 and E5, the specific silicic acid uptake rates (V, h−1)defined as the silicic acid uptake rate per unit BSi, wereplotted as a function of the added DSi concentrations(Fig. 7). The maximal specific uptake rate at infiniteDSi concentrations (Vmax, h

−1) and the half saturationconstant (KS, μM) defined as the DSi concentration atwhich V=Vmax /2, were determined by fitting the datawith the non-linear method of Wilkinson (1961). Thehalf saturation constant, KS, increased from E2 to E5with values of 1.6 (±0.2), 3.3 (±0.3) and 5.6 (±0.9) μMin E2, E4 and E5, respectively (Fig. 8). An increase wasalso observed for Vmax with values ranging from 0.009(±0.000) to 0.017 (±0.001) h−1.

4. Discussions

4.1. Phytoplankton composition

After a few days, and despite a dominance ofnanophytoplankton at the beginning of the experiment,all enclosures were rapidly dominated by microphyto-plankton as evidenced by the particulate organic nitrogen

Fig. 8. Relation between KS values and Si fertilization rates.

Fig. 7. Concentration dependence of silicic acid uptake rate by naturaldiatom assemblages in (a) E2 on day 7, (b) E4 on day 11 and (c) E5 onday 11. Data points show the measured values of the specific uptakerate (V). The curve represents the Michaelis–Menten hyperbola fittedto the data by the non-linear regression method of Wilkinson (1961).For the three experiments, a significant correlation ( pb0.01) wasmeasured between the data and the model.



(PON) fraction N10 μm that increased from almost 25%of the total PON on day 1 to more than 80% on day 9.Among microphytoplankton, diatoms (mainly C. debilis)largely outgrew non-siliceous species in all enclosures(Fig. 5c) as confirmed by the significant correlationmeasured between BSi and the N10 μm PON concentra-tions (Fig. 4). In addition, the calculated slope betweenthe two variables (0.85) was highly comparable to themean Si:N ratio of 0.82 measured for Chaetoceros spp.(Brzezinski, 1985) which is the major genus of themicrophytoplankton in all enclosures. Chaetoceros hasalready been observed in spring in the Bay of Brest(Beucher, 2003) and represented up to 50% of the totaldiatom cell counts in the Bay in March and April 2004(Beker, pers. com.).

Environmental conditions (light, temperature, turbu-lence…) were probably particularly favourable to diatomdevelopment, but their dominance may also have beenpartly controlled by zooplankton dynamics within theenclosures. Indeed, the zooplankton community wasmainly composed of copepods and appendicularians(O. cf. dioica); appendicularians being the dominanttaxon at the end of the experiments in all enclosures.Appendicularians are known to graze preferentially onnano- and picoplankton while copepods can hardlycapture particles of this size and graze preferentiallyon larger cells such as diatoms (Fernández et al., 2004).In addition, several studies have highlighted the pred-ator–prey interactions between calanoid copepods andappendicularians. For example, Stibor et al. (2004)observed that copepods feed on appendicularian eggsand larval stages and therefore control appendicularianabundance. These authors examined the influence ofdifferent initial copepod density on the population dy-namics of O. dioica and considered a density of about10 copepods L−1 a critical level below which appendi-cularian may bloom. In our study, the maximal copepodabundance did not exceed ∼2 individuals L−1, whichmay explain the development of O. cf. dioica. In ad-dition, the rapid development of O. cf. dioica whilecopepods density remained almost unchanged was notsurprising knowing that copepods have generation timesof several weeks in temperate waters while appendicu-larians have much shorter generation times, in the orderof several days (Uye and Ishino, 1995). Also, theincrease in the appendicularian density from enclosureE1 to E6 is coherent with the results of Stibor et al.(2004) and Vadstein et al. (2004) who observed thatnutrient addition had a positive effect on appendicu-larians as it stimulated the production of algal food.Finally, the diatom dominance may result from both astrong grazing pressure of O. cf. dioica on nanophyto-

plankton and a low grazing pressure of copepods ondiatoms.

4.2. Diatom dominance and silicic acid availability

During our experiment, the initial DSi concentrationwas high enough to allow diatom dominance, even inthe unfertilized enclosure E1. However, after a few days,DSi was depleted and Si:N:P ratios indicated that Si wasthe most potentially limiting nutrient over the time scalestudied, whatever the enclosure (Fig. 1c). Although DSidepletion may induce a shift from diatoms to non-siliceous species (Officer and Ryther, 1980; Smayda,1990), C. debilis rapidly dominated the phytoplanktoncommunity to represent 75±15% of the microphyto-plankton cell numbers at the end of the experiment.Initial conditions obviously affected the structure andthe evolution of the population but it is also evidentthat daily Si fertilizations were strongly involved in themaintenance of diatom dominance after DSi depletion.In the unfertilized enclosure E1, diatom dominance waspresumably based on the dissolution of diatom frustulesin the water column. This dissolution hypothesis wasthe most plausible hypothesis to explain the mainte-nance of diatoms in the control for the entire 3 weeks.It should be noted that the highest fertilization rate(4 μM Si L−1 d−1) that was determined to be fourtimes the maximal benthic flux measured in the Bay ofBrest, was not high enough to avoid DSi depletion inE6. This result suggested that a higher fertilizationrate could have induced a higher diatom concentrationin E6.

Egge and Aksnes (1992) showed that diatoms alwaysnumerically dominated the phytoplankton communitywhen DSi concentrations were above 2 μM. Belowthis value, they reported that diatom dominance ceasedor became more variable. As this value of 2 μM wasdetermined using data collected under a wide range ofenvironmental conditions, it is now commonly used as anutrient criterion to predict diatom dominance in eithermesocosm experiments or marine ecosystems. Thisstudy provides a completely different scheme sincediatoms strongly dominated microphytoplankton at theend of the experiment when DSi depletion wasobserved. The combination of our results and similarobservations of diatom dominance in DSi concentra-tions under 2 μM between 1998 and 2001 in the Bay ofBrest (data not shown), confirm that the residual DSiconcentration of 2 μM should not be considered as areliable criterion to predict diatom dominance in marineecosystems and suggest that emphasis should be givenmore to nutrient loading.



4.3. Response of diatoms to fertilization rate

Increase in the diatoms' Si uptake rates to rising DSiconcentrations provided a direct evidence of Si limi-tation. The half saturation constant, KS, measured inE2 (1.6±0.2 μM) was similar to the values measured inthe Bay of Brest (1.3–2.1 μM; Ragueneau et al., 2002).The KS were higher in E4 (3.3±0.3 μM) and E5(5.6±0.9 μM), but remained in the range of valuesreported for natural diatom assemblages (Nelson andTréguer, 1992; Claquin et al., 2006). The 3-fold increaseof KS between E2 and E5 could be due to a change incell size (Raven and Kubler, 2002) or an adaptation tovarious environmental factors (Dugdale and Wilkerson,1998; Leynaert et al., 2004; Sarthou et al., 2005).

It has been demonstrated that a given species mayrapidly reduce its cell size under nutrient stress in a fewgeneration, leading to an increased surface-to-volumeratio, thereby increasing the cells' ability to take upnutrients (Raven and Kubler, 2002; Leynaert et al.,2004). This effect has been reported for various limitingfactors, both in the field and in cultures. For example,cell size of Stephanodiscus minutulus, a freshwater dia-tom, was significantly smaller for Si-, N-, and P-limitedcultures than for non-limited cultures (Lynn et al.,2000), whereas in the Jiaozhou Bay (China), in situ Silimitation led to a shift from large to smaller diatom cells(Shen, 2001). In our study, over the 3 week duration ofthe experiment, no significant change in the cell size thatcould explain the KS variation was observed (B. Beker,Marseille, pers. com.).

Although no obvious relationship has ever beenevidenced, we generally expect the KS to be higher athigh ambient DSi concentrations and lower at low DSiconcentrations (Claquin et al., 2006). As an example,it has been shown that in quasi-stationary state sys-tems, in situ diatom populations were able to regulatetheir KS to the ambient DSi concentration (Dugdaleand Wilkerson, 1998). In our experiment, ambient DSiconcentrations at the time of sampling for the kineticsexperiments were relatively low compared to the KS

values (1.1, 0.4 and 0.1 μM Si(OH)4 in E2, E4 and E5respectively) but we observed that the half saturationconstants increased with the fertilization rate (Fig. 8).This result suggests that diatoms did not regulate theiraffinity to the ambient DSi concentration but to thefertilization rate.

It has been demonstrated that diatom cells can reg-ulate Si uptake by controlling the amount, the localisa-tion or the type of Si transporter (SIT) on its surface(Hildebrand et al., 1998). For example, five differenttypes of SITs were characterized in C. fusiformis

(Hildebrand et al., 1998), three others in T. pseudonana(Armbrust et al., 2004) and several others in a number ofdifferent species (Thamatrakoln et al., 2006). All theseSITs may have different affinities for transport (Hildeb-rand, 2000; Hildebrand and Wetherbee, 2003). Therelation between KS and fertilization rate may be theconsequence of such a regulation by C. debilis in res-ponse to a change in the fertilization rate. These resultssupport the hypothesis of an adaptation of C. debilis toDSi addition in this study.

As for the KS, the maximal uptake rate, Vmax, in-creased with fertilization rate from 0.009 (±0.000) h−1

to 0.017 (±0.001) h−1. This increase of both KS andVmax, while all enclosures exhibited a dominance ofC. debilis, supports the findings that diatoms may pres-ent some degree of flexibility in acclimating to DSiavailability (Olsen and Paasche, 1986). In particular,these results indicate that Si uptake parameters, insteadof being species-specific constants, are rather suscepti-ble to variation within the same species, in response todifferent environmental conditions. The large range ofour experimental KS values (from 1.6 to 5.6 μM), com-pared with in situ values (from 1.3 to 2.1 μM) measuredin the Bay of Brest for Chaetoceros spp. (Ragueneauet al., 2002), confirmed that KS was highly sensitive tothe fertilization rate. The Vmax increase also suggestedan influence of nutrient additions, but the range ofvariation remained very small compared to in situ val-ues in the Bay of Brest (from 0.040 to 0.160 h−1) andto values in the literature for the natural environmentor in culture experiments (Martin-Jézéquel et al., 2000;Sarthou et al., 2005; Claquin and Martin-Jézéquel,2005). As mentioned previously, changes in KS maybe the consequence of changes in transporter affinity,whereas modifications of Vmax follow the variation inthe number of transporters. In our study Vmax appearedto be less sensitive than KS to fertilization rate, whichmay indicate a lower flexibility in this parameter inC. debilis under such growth conditions.

5. Conclusion

This study showed that quantitative modifications ofthe fertilization rate in early spring (nutrients ratio waskept constant in this study) only changed the magnitudeof the bloom since all enclosures presented a strongdominance of C. debilis after a few days. These resultswere surprising considering that the intensity of Sirecycling at the sediment–water interface was supposedto be strongly involved in this diatom dominance(Ragueneau et al., 2002). As diatoms dominate evenat low fertilization rate, it raised questions about the



impact of the removal of C. fornicata on phytoplanktonsuccession and the possible development of harm-ful algal blooms. The analysis of the data from twomesocosms experiments performed in June and Sep-tember 2004 will further our understandings of thereal influence of benthic flux change over the wholeproductive period.

The distinct trends that were found at the end ofthe experiment between the highly enriched enclosuresE5 and E6 and the others suggested that a proliferationof C. fornicata is more likely to affect phytoplanktondynamics. In E1 to E4, both the PON and the BSiincreases tended to stabilize at a maximal value after afew days. To the contrary, the increases of PON and BSiin E5 and E6 were followed by a net decrease at the endof the experiment that indicated a massive sedimenta-tion of cells. It seems that fertilization rates exceeding athreshold of 1 μM Si d−1 are likely to enhance sedimen-tation processes. Such processes could deeply perturbthe benthic communities as massive sedimentation ofphytoplankton cells or aggregates can induce anoxicconditions or gill clogging. In the Bay of Brest, themaximal benthic flux of ca 1.2 μM Si d−1 was mea-sured in spring on a site with a density of C. fornicataN1000 individuals m−2 (Martin, 2005). Knowing thatthe mean density of C. fornicata in the whole Bay ofBrest is of ca 260 individuals m−2 (Guérin, 2004), wecan concluded that the mean DSi benthic flux measuredin the Bay is still far from the perturbation threshold of1 μM Si d−1.

Acknowledgements

This research was funded by the Région Bretagneand is part of an INVABIO 2 project that was fundedby the Ministère de l’Ecologie et du DéveloppementDurable. We especially thank the Lanvéoc City Hall, V.and D. Dham, B. Beker, A. Saget and the “Bar de laCale” for technical assistance. [SS]

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