OBJECTIVE 2.

PRODUCTION AND FATE OF ORGANIC MATTER IN CONTRASTING OLIGOTROPHIC ENVIRONMENTS

The major objective is to study how production, mineralisation and export of organic matter is depending on the community structure in contrasted oligotrophic areas. We will quantify the flow of material (biogenic elements) through each ecosystem and particularly focus on the coupling/uncoupling between carbon and nutrient supply and removal processes.

Objective 2.1. Characterization of nutrient availability

 Measurements of nutrient (N, P, Si, Fe) concentrations and/or availabilities

Nutrient availbility in the photic zone controls primary production in the organic matter, and thus exerts a strong influence on the species composition of the food web. It is dependant on several inputs, recycling and export fluxes. Nutrient concentration in the upper layer represents the equilibrium state between all those fluxes and is the first indicator of nutrient availability. Nevertheless, as nutrient concentrations are very low in marine oligotrophic habitats, these measurements are very difficult and represent a challenge . Nanomolar methods for nitrate and nitrite concentrations will be used. Continuous profiling of nitrate using an in situ ultraviolet sensor (Johnson & Coletti, 2002) will enable us, for the first time, to study small time scale variability in the nitracline. We will study dissolved inorganic phosphate availability, probably the principle factor controlling planktonic populations especially the diazotrophes ( A 2.1), and the N:P balance in the euphotic layer (A 2.2) using methods recently adapted to ultra oligotrophic conditions (Rimmelin & Moutin, 2005, Tanaka et al., submitted, Thingstad & Mantoura 2005). Silicic acid availability ( A 2.3) may also play a central role in controlling the export production (Leblanc et al., 2003). High dissolved iron concentrations have previously been observed in the western Mediterranean Sea at the end of the stratified period (Guieu et al. 2002, Bonnet & Guieu submitted , Sarthou & Jeandel, 2001). However, no data on iron availability ( A 2.4), which may exert a strong control on N 2 fixation rates (Kustka et al., 2002), are yet available for the eastern Mediterranean Sea.

 Identification and quantification of the major external biogeochemical fluxes related to nutrient (N, P, Si, Fe) availability

New production, supported by external sources of nutrients into the photic zone, may be quite low in oligotrophic habitats but is non - the- less a crucial fraction of total autotrophic production. Indeed, over a long term basis, there is an essential equilibrium state between New Production and export from the photic layer, to maintain the organic production in that layer.

 Input by hydrological process

Nutrient flux at the bottom of the photic layer is the result of permanent and/or intermittent advective or diffusive effects. It may be calculated as the product of an eddy diffusion coefficient by a nutrient concentration gradient. This flux is not well established in oligotrophic areas and is a current subject of study (Lévy 2003, McGillicudy & Robinson 1997, Balter et al. 2005, and comments from Levy 2005). We will study sites where low potential advection occurs to minimize lateral input, for example in the centres of anticyclonic gyres. Nevertheless, advection will be evaluated. Vertical nutrient gradients are easy to measure but this is not the case for eddy diffusion coefficients (K z ). These can however, be measured using specific methods based on correlations between vertical velocity fluctuations and temperature or salinity (Lewis et al. 1986). Unfortunately, these methods are expensive and take a long time to perform. Hence the eddy diffusivity is most often estimated using indirect methods based on fine-scale measurements (CTD and ADCP). One method currently used, relies on the evaluation of the Richardson number ratio of the vertical gradient of density per mass unit on the squared vertical shear of the horizontal current. This number is involved in Kelvin Helmotz instabilities that are probably responsible for the major vertical nutrient flux in stratified areas (Gregg 1987, Large et al. 1994, Kantha & Clayson 1994). An accurate measurement of this number can be obtained using a CTD SBE 911 coupled to a LADCP mounted on the rosette. Another method using the ratio of vertical temperature or salinity gradient variances, over a small to medium scale (10 cm and 5 m for example), could also be used. Simultaneous microstructure measurements that resolve the dissipative scales will be performed using a full ocean depth un-tethered profiling system, “VMP5500”. This profiler is equipped with microsensors for temperature and shear that enable two independant accurate estimates of Kz. These two estimates will be used to test the relevance of the different indirect methods based on classical CTD and ADCP measurements. This validation of indirect estimates of Kz will then be used as a guide to estimate Kz when microstructure measurements are not carried out.

 Atmospheric input – N 2 fixation

Aeolian dust transport represents, on a global scale, the dominant source of iron, an essential micronutrient for phytoplankton growth, to the ocean (Duce and Tindale 1991; Gao et al, 2001; Jickells et al., 2005) . Moreover, it has been shown that the Saharan aerosols can also represent a significant source of phosphate (Ridame & Guieu 2002) . Consequently, Saharan dust inputs could stimulate in situ biological productivity in oligotrophic ecosystems, such as the Mediterranean Sea and alter community structure, distribution of nutrients and finally the net sequestration of atmospheric CO 2 . Considering the short duration of our survey (4 or 5 days at each station) and the low frequency of atmospheric nutrient input by aerosols, these inputs will probably be negligible and will not be considered. We will only focus on nitrogen input by nitrogen fixation, which may be a major source of new nitrogen in the eastern and western Mediterranean Sea (A 2.5), and study the influence of atmospheric dust deposits on the food web and their potential impact on nitrogen fixation, using microcosm experiments (A 2.6). Pantoja et al. (2002) estimated that up to 20% of the nitrogen in the western basin and up to 90% in the eastern basin may be derived from biological N 2 fixation, but there is very little data available for the eastern Mediterranean Sea.

Specific questions: Is nitrogen fixation a major source of new nitrogen for primary production in the photic layer? Do direct measurements of eddy diffusion coefficients reasonably agree with previous estimations?

Objective 2.2. Organic matter production and food web structure

Because of light/energy requirements, marine organic matter is almost entirely produced in the upper photic zone of the ocean. Variable primary production fuelled by physical processes acting over a wide range of scales, interact with predation to define the species composition of planktonic populations (Gargett & Marra, 2002). The basic principle of the conceptual food web structure, which has not been critically challenged since its original description (Johannes, 1965; Thingstad et al. 1999), is to consider two trophic strategies (Fig. 4). Osmotrophy refering to organisms that feed by taking up dissolved nutrients and phagotrophy refering to organisms that feed by eating particulate matter. Osmotrophs include both the heterotrophic bacteria and autotrophic phytoplankton, while the predatory food chain includes both the protozoa, mesozooplankton and higher predators (all heterotrophs).

Fig. 4. Idealized food web model for the photic zone and biogeochemical fluxes of nutrients and carbon (redrawn from Thingstad et al. 1998 & 2005)

 Biogeochemical fluxes in relation to osmotrophic production

The objectives are to measure fluxes of biogenic elements (including carbon) and to determine parameters that will help to represent these fluxes (P vs I parameters, K s , V max , affinity constants…). Classical approaches using stable ( 15 N, 13 C) and unstable ( 14 C, 33 P, 32 Si) isotopes for measuring biogeochemical fluxes will be used, along with new techniques that can help to address by which species (or group of species) a specific element has been taken up. We will study the dissolved organic carbon primary production (A 2.7), recently acknowledged to significantly contribute to the total primary production in oligotrophic areas (Marañón et al., 2005). We will quantify nitrification as well as the impact of dissolved organic nitrogen excretion on the measurement of "new" nitrate uptake (A 2.8). New methods concerning both dominant species separation (Lebaron et al 2001, Servais et al 2003) and low detection limits of chemical analyses (Rimmelin & Moutin, 2005; Duhamel et al., in press.) will allow to define new specific uptake parameters. We will characterize the dissolved inorganic phosphate uptake in a group of species at a specific level (A 2.9) and define the biochemical fate ( A 2.10). This will give us a detailed view of the overall P-requirements for plankton growth (Van Mooy, 2003) . The silicification process in marine diatoms (A 2.11) will be investigated using a new labelling technique (Shimizu et al., 2001; Leblanc & Hutchins, 2005) that enables us, for the first time, to discriminate between active and non-active siliceous biomass.

 Phagotroph production (secondary production)

The planktonic food web structure influences the fate of primary production in the ocean and has consequences for the CO 2 transfer process. It is generally understood that when small eukaryote or prokaryote bacteria dominate the microbial community, the grazers of picoplankton are small protozoa which do not produce rapidly sinking faecal pellets. Despite their omnipresence and their pivotal role in the energy flow in marine waters on a global scale, the physiological ecology of these organisms is poorly understood (A 2.12). Several grazing steps are necessary to enable primary production to be incorporated into the upper trophic levels. Therefore, most of the carbon fixed by phytoplankton is respired and remineralised by the microbial community in the surface mixed layer and there is little or no net uptake of CO 2 from the atmosphere to the sea. In contrast, when large phytoplankton cells dominate, they can either be deposited to the bottom or be grazed by copepods and other mesozooplankton which produce rapidly sinking faecal pellets. However, Mesozooplankton, i.e. copepods have recently been coupled to lower trophic levels in the eastern Mediterranean Sea (Thingstad et al. 2005) and their contribution to the carbon cycle of oligotrophic areas needs to be reconsidered ( A 2.13). The upper meso- and macrozooplankton , largely under-sampled by classical techniques, may also significantly contribute to the vertical transport of organic matter ( A 2.14).

 Biogeochemical process from optical measurements

The use of optical measurements for inferring biogeochemical process on a diel scale is now well acknowledged (Siegel et al., 1989; Claustre et al., 1999). The technique for deducing high frequency particulate organic carbon from attenuation measurements (diffusion) has revealed higher estimates for primary production in the oligotrophic environment (Claustre et al., in prep.). Most of these studies have been performed using the attenuation coefficient of suspended particles in the red part of the spectrum (equivalent to the particulate scattering coefficient, b p ), combined with chlorophyll-fluorescence measurements. The simultaneous measurements of b p , b bp (sub-micrometer particles) and of the Chl-fluorescence signal may be used to analyse the diel scale of the particulate organic matter (Loisel et al., 2002; Behrenfeld et al., 2005) and to understand how the diel variations of sub-micrometer and mostly non-living particles, as revealed by b bp , behave compared to those of the larger and mostly authotrophic particles, as revealed by b p . ( A 2.15). Over the diel cycle and between ocean regions of changing nutrient stress, physiological separation of photosynthetic processes (initial charge separation and carbon fixation.) can cause a severe decoupling between variable fluorescence and carbon fixation patterns. The understanding and characterization of such decoupling (A 2.16) constitutes the basis for relating high-resolution (even space-based) fluorescence data to carbon cycling in the sea.

General questions: What are the characteristics of the dominant species concerning photosynthesis, nutrient uptake and phagotrophy ?

Objective 2.3. Organic matter mineralization and food web structure

The question of specific mineralization of each element (carbon and nutrients) is central because differential mineralization may lead to decoupling of biogeochemical cycles within the water column (Raimbault et al., 1999; Karl et al., 2001, Leblanc et al. 2003 ). It is generally believed that phosphate is more rapidly recycled than nitrogen which is more rapidly recycled than silicic acid. Such parallel recycling flux measurements are scarce and the latter assumption is essentially derived from indirect measurements. The study of organic matter degredation will focus on the relationship between mineralization and bacterial diversity ( A 2.17). Hence, examining the relationship between diversity and functionality within the bacterial community (ectoenzymatic activity, uptake of specific organic compounds representative of a chemical family) is a major challenge for understanding the impact of prokaryotic heterotrophic processes on mineralization of organic matter along the water column . We will also study the factors controlling bacterial production and the consequences of such controls on heterotrophic activity ( A 2.18), the hydrolysis rate of Dissolved Organic Phosphate ( A 2.19) .

General questions: What are the changes in prokaryotic heterotrophic activity and community structure in relation to horizontal (west-east) and vertical (surface to depth) nutrient gradients and the composition of the dissolved organic matter?

Objective 2.4. Organic matter export (particulate and dissolved matter)

The structure of pelagic ecosystems influences the size and type of downward flux of biogenic carbon in the sea (Legendre and Rassoulzadegan, 1996). This vertical flux is composed mainly of large particles such as fecal pellets, hard parts of zooplankton, amorphous aggregates, marine snow (Fowler and Knauer, 1986; Silver and Gowing, 1991), and senescent diatoms, particularly in the aftermath of blooms (Billet et al., 1983). Fecal pellets can sink quickly: 20-900 m d -1 for copepods (Lorenzen, 1983; Welschmeyer and Lorenzen, 1985), and up to 2700 m d -1 for large gelatinous zooplankton (Bruland and Silver, 1981; Madin and Purcell, 1992). High sinking velocities can lead to the efficient export of biogenic carbon. The downward flux of biogenic carbon can therefore be largely dominated by carbon of zooplankton origin (Thibault et al ., 1999).

We will study the C/N/P/Si stoichiometry of settling particulate matter ( A 2.21) because despite its biogeochemical relevance, there are few direct measurements available (Geider & La Roche, 2002). We will also examine the role of larger particulate matter responsible for the marine snow rarely observed in oligotrophic areas ( A 2.22) and quantify instantaneous DOC export.

Objectif 2.5. Oxygen budget

The balance between photosynthesis and respiration in the microbial community defines the metabolic state of the studied ecosystem (Net autotrophic or heterotrophic) and its capacity to export carbon. The budget on a total oceanic scale is uncertain and the area of greatest uncertainty lies paradoxically in the surface layer of the ocean (Williams, ASLO meeting 2006). The originality of the approach used will consist of comparing the variations in oxygen concentration over different time scales, in situ and in incubation bottles, using a new system called a "Productivity Autosampler" (A 2.23). We will also use the more classical technique for assessing the daily budget (Gross and Net Community Production, community respiration), using the nychthemeral variations in oxygen concentration. This will yield information on the respiration rates of the autotrophic and heterotrophic communities (Pringault et al. L&O method, submitted) enabling us to determine the impact of the microbial community on the carbon cycle.

Objective 2.6. Responses in the microbial food web to pulse nutrient addition (microcosms studies)

Phosphate addition to the P-starved and ultraoligotrophic surface water of the Cyprus Gyre, Eastern Mediterranean in a Lagrangian experiment caused unexpected ecosystem responses (Thingstad et al. 2005). The system exhibited a decline in chlorophyll and an increase in bacterial production and copepod egg abundance. The results were supported by those obtained from an on-board microcosm experiment during the Lagrangian experiment (Zohary et al. 2005). Although N and P co-limitation hindered phytoplankton growth, they explain that P may have been transferred through the microbial food web to copepod via two, not mutually exclusive, pathways: (1) bypass of the phytoplankton compartment by P uptake in heterotrophic bacteria and (2) tunneling, whereby P luxury consumption rapidly shifts the stoichiometric composition of copepod prey (Thingstad et al. 2005). These mechanisms are not fundamentally new for limnologists but have been explored to a very limited extent in marine systems. It is thus necessary to test whether these mechanisms exist and how pulsed P addition is used by the microbial food web and mesozooplankton in different P limited regions. Since the result from the on-board microcosm experiment was fairly similar to that from the in situ experiment (Thingstad et al. 2005; Zohary et al. 2005), microcosm experiments (A 2.24) will allow testing the above mechanisms. In addition, microcosm enrichment experiments will be conducted on board using trace-metal clean techniques to address in particular the role of Fe and dust on N 2 fixation at a selected number of sites on water sampled in the sur face mixed layer.

Studies driven at the scale of hour to year in oligotrophic environments have shown that most of the nutrient inputs from the deep to the surface layers were attributed to punctual pulses (Dickey et al 2001). However the consequences of such pulse of nutrient have been scarcely studied . Atmospheric input are also punctual and thus, it is particularly interesting to study simultaneously the in situ ecosystem in its most probable steady state as well at its response after a nutrient input.

Specific questions: What are the consequences of pulsed nutrient addition on the microbial food web and mesozooplankton in marine waters with different oligotrophic status? What are the respective role of Iron and phosphate on the control of the N 2 fixation rates?