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International audience; Over the last decades, mass developments by the filamentous conjugating green alga Mougeotia have been followed in three large peri-alpine lakes (Lake Geneva, Lake Garda, Lake Maggiore) and in the sub-tropical Lake Kinneret. The aim of this study is to highlight annual and interannual patterns of Mougeotia biomass in the studied lakes and select key environmental parameters that may favour and maintain its mass development. Our results confirm former studies that planktic Mougeotia favours meso-oligotrophic conditions and becomes dominant when annual mean total phosphorus concentrations in the epilimnion fall below 20 A mu g l(-1). This triggering factor has effect with interactions of other environmental circumstances such as the water column stability. Physiological and morphological features of the taxon make it a successful competitor under stratified conditions. Results also showed that in three out of the four studied lakes, the annual peak was higher when the annual population development started earlier. Focusing on Lake Geneva, depth and strength of the thermocline, as well as wind speed in the beginning of summer that can cause nutrient replenishment and mix the epilimnion are key factors in the blooming of the taxon.

Heterotrophic bacteria contribute significantly to food web structure and biogeochemical cycling in the ocean. In oligotrophic regions such as the Mediterranean Sea, the functioning and productivity of the pelagic ecosystem largely depends on the activity of heterotrophic bacteria which are at the center of the balance between exported and mineralized carbon. As a result of pioneer studies developing techniques to assess bacterial production and abundance, these parameters have been measured in the Mediterranean Sea since the late 1980s. Much more recently, thanks to the advances in molecular methods for exploring microbial biodiversity, an understanding of the biogeochemical implications of the assemblage's taxonomic composition begins to emerge. Today, research efforts on the role of heterotrophic bacteria in the Mediterranean Sea face new challenges related to understanding and foreseeing the probable responses to changing natural and anthropogenic forcing. This chapter aims at reviewing the current state of knowledge on the variability of bacterial abundance, activity and community structure at both spatial and temporal scales in the pelagic waters of the Mediterranean Sea. In addition, the response of heterotrophic bacteria to major human driven changes is evoked. Finally, future directions dealing with key questions and new tools related to research on heterotrophic bacteria are proposed.

During May 2001 and May 2002, the structure and function of the microbial community within and outside the Cyprus quasi-stationary warm-core eddy in the Levantine Basin of the eastern Mediterranean was studied down to the depth of the bathypelagic layer. We present here the detailed description of the microbial food web in one of the most oligotrophic and P-starved marine systems on earth. The isothermal layer was at the depth between 20 and 260/300m at the core of the eddy, and between 20 and 100/110m outside. Nitrate and phosphate were found at higher concentration between 100 and 500/800 m outside the eddy compared within the core of the eddy, but the vertical diffusive flux of nitrate and phosphate across the pycnocline was higher within the core of the eddy. There were only minor differences in microbial abundance in the euphotic, layers of the two sites. It is suggested that the differences in the areal supply of nutrients to the isothermal layer, between the two sites, resulted in essentially a similar volumetric supply of nutrients to the euphotic layer. This suggests that the results of this study can be applied to describe the microbial food web within the euphotic layer over the larger area of the Levantine Basin, which exhibits ultra-oligotrophic and P-starved conditions. Primary production and abundances of the microbial community were somewhat higher in May 2001 than in May 2002, possibly because of higher nutrient fluxes in the euphotic layer, which are probably the result of deeper winter mixing in 2001, although a later onset of winter mixing or increased dust supply could not be discounted. In the euphotic layer, heterotrophs (bacteria, heterotrophic nanoflagellates (HNF), and ciliates) dominated (60-70%) the microbial carbon biomass. Heterotrophic ciliates were found to be much more abundant in the upper 50 m of the water column, while no consistent pattern wasfound for bacteria and HNF throughout the euphotic layer. Autotrophs showed a maximum distribution at the deep chlorophyll maximum found between 100 and 130m. In the euphotic layer, the relationships between biomass and production for phytoplankton and bacteria suggested a higher top-down control on the phytoplankton in the upper similar to 50 in and a consistently tight top-down control on the bacterial biomass throughout the euphotic layer. The phosphate addition experiment in the Cyprus Eddy suggests that the close predator-prey relationships within the microbial heterotrophic community were required for the rapid transfer of a limiting element to higher trophic levels without biomass oscillations in the P-fertilized surface mixed layers (0-20m). The results from the unmodified system in this study suggested that the rapid P transfer mechanisms would function only in the upper similar to 50m, while the element transfer would be based on a predator-prey relationship with more conceivable biomass oscillations in the deeper waters (similar to 100-160m) of the euphotic layer. In the Mediterranean Sea, nutrient concentrations, POC export, and integrated chlorophyll and primary production all tend to decrease toward the east. Our results together with a literature survey showed that abundances of the microbial components in the euphotic layer were not consistently lower in the study area than in the northwestern Mediterranean and that abundances of bacteria and HNF found in the mesopelagic and bathypelagic layers of the study area were within the reported ranges and quite similar to those found in the northwestern Mediterranean. This suggests that the oligotrophic status and the lw export production are not reflected in the abundance of the microbial components down to the bathypelagic layer of the eastern Mediterranean. (C) 2007 Elsevier Ltd. All rights reserved.

With no requirement for synoptic treated (IN) and control (OUT) stations, analysis of covariance is an interesting statistical technique for testing IN-OUT differences in Lagrangian experiments, but it has inherent limitations due to its assumption of linear responses. With this limitation properly considered, we find that analysis of covariance strengthens, not weakens, experimental support for the food-web transfer mechanisms we proposed.

Phosphate (P), nitrate (N) or P+N added in a microcosm experiment to oligotrophic waters of the Eastern Mediterranean influenced by near-shore waters triggered a range of responses in the autotrophic and heterotrophic compartments of the system. Chlorophyll a increased in all treatments, including the no-addition control, implying that nutrients became available also from internal sources (recycling). Larger and faster biomass increase as well as a larger P utilization took place in the P+N treatments. Diatoms bloomed in the P+N treatments whereas coccolithophores bloomed following the addition of P ultimately reaching N-limitation. Bacterial activity responded with a transient peak to both low P-alone and N-alone additions (0.01 and 1 mu M, respectively). For reasons not well understood, no such response was observed at higher P-alone additions (0.05 and 0.5 mu M), whereas at the two highest P+N additions the positive response was delayed. We therefore were unable to conclude conclusively on bacteria] limitation. In most cases, the increase in bacterial activity was not matched by an increase in abundance, suggesting a tight top-down control of the biomass. Instead, heterotrophic nanoflagellate and ciliate abundances increased in all treatments. A slightly elevated orthophosphate turnover-time (T-t) (32h) in the initial waters did not give a clear indication of P-limitation, although the system could absorb the lowest P-addition (0.01 mu M) without increase in T-t N alone lead to a reduction in T-t as would be expected in an N-limited system consuming existing surplus P after N-addition. The response of the near-shore influenced system used in this study was in accord with the `classical' response to nutrient introduction-increase in chlorophyll a and in large size phytoplankton. In contrast, in the ultraoligotrophic Cyprus Eddy [Krom, Thingstad, Carbo, Drakopoulos, Fileman, Flaten, Groom, Herut, Kitides, Kress, Law, Liddicoact, Mantoura, Pasternak, Pitta, Polychronaki, Psarra, Rassoulzadegan, Skjoldal, Spyres, Tanaka, Tselepides, Wassmann, Wexels-Riser, Woodward, Zodiatis, Zohary, 2005. Overview of the CYCLOPS P addition lagrangian experiment in the Eastern Mediterranean. Deep-Sea Research II, this volume.], the short T, (< 4h) indicated P-limitation, the combined addition of P and N (as ammonium) induced a bloom of picocyanobacteria [Zohary, Herut, Krom, Mantoura, Pitta, Psarra, Rasssoulzadegan, Stambler, Tanaka, Thingstad, Woodward, 2005. P-limited bacteria but N&P co-limited phytoplankton in the Eastern Mediterranean-a microcosm experiment. Deep-Sea Research II, this volume.] and the in situ P alone addition led to a decrease in chlorophyll. (c) 2005 Elsevier Ltd. All rights reserved.

The response of the microbial food web to P-addition was studied during a 10-day Lagrangian experiment in the Eastern Mediterranean during which orthophosphate was added to the surface water of the Cyprus anticyclonic eddy. Very low levels of all microbial populations (heterotrophic bacteria, Synechococcus, Prochlorococcus, autotrophic and heterotrophic nanoflagellates, ciliates) and bacterial production were detected, verifying the extreme oligotrophic character of this area. The microbial biomass was dominated by heterotrophs and the heterotroph/autotroph ratio increased from 1.05 before to 2.8 after the P-addition, on day 4. Bacteria took advantage of the supply of the putative limiting factor (P) increasing their production but not their abundance. The heterotrophic nanoflagellates remained stable in numbers. By contrast, Synechococcus and autotrophic nanoflagellates decreased after the P addition. This is an indication of consumption by ciliates which were the only organisms that showed a significant increase in abundance during the first 4 days after the P addition, relative to the period before the addition as well as to the second phase of the experiment (days 5-9). In particular, the mixotrophic ciliate biomass increased by 50% after the P release. In environmental conditions of general resource scarcity as is the case of the Eastern Mediterranean, the addition of the putative limiting factor (P) was reflected in the increased abundance of microzooplankton shortly after the enrichment as a result of a quick transfer of energy (''heterotrophic by-pass'') through bacteria and heterotrophic nanoflagellates. A ``mixotrophic by-pass'' of phytoplankton primary producers also occurred, transferring the P-addition driven primary production to higher trophic levels through mixotrophic ciliates. (c) 2005 Elsevier Ltd. All rights reserved.

CYCLOPS was a European Framework 5 program to further our understanding of phosphorus cycling in the Eastern Mediterranean. The core of CYCLOPS was a Lagrangian experiment in which buffered phosphoric acid was added to a < 4 x 4 km patch of water together with SF6 as the inert tracer. The patch was followed for nine days in total. Results obtained prior to the experiment showed that the system was typically ultra-oligotrophic and P-starved with DON:DOP, PON:POP and DIN:DIP all having ratios greatly in excess of 16:1 in surface waters. To our surprise, we found that although the added phosphate was rapidly taken up by the microbial biota, there was a small but significant decrease in chlorophyll a and no increase in primary production, together with an increase in heterotrophic bacterial activity, ciliate numbers and in the gut fullness and egg numbers in the zooplankton community. A microcosm experiment carried out using within-patch and out-of-patch water showed that the phytoplankton community were N and P co-limited while the bacteria and micrograzers were P-limited. Thus this system tends to N and P co-limitation of phytoplankton productivity in summer possibly caused by bioavailable DIN being converted into non-bioavailable forms of DON. On the basis of the data collected within the programme it was concluded that this behavior could be explained by three non-mutually exclusive processes described as (1) trophic by-pass in which the added phosphate gets directly to the grazing part of the predatory food chain from the heterotrophic bacteria bypassing the phytoplankton compartment phosphate, (2) trophic tunnelling in which phosphate is rapidly taken up by both phytoplankton and bacteria via rapid luxury consumption. This causes an immediate change in the phosphorus content but not the abundance of the prey organisms. The added P then ``reappears'' as responses at the predator level much more rapidly than expected, and (3) mixotrophic by-pass in which inorganic nutrients, including the added P, are taken up by mixotrophic ciliates directly, bypassing the phytoplankton. For details of the results of this study and the processes described, the readers are referred to the relevant papers within this volume. The implications of these results for nutrient cycling in the Eastern Mediterranean are discussed. In particular it is noted that the efficient and rapid grazing observed in this study might explain why the system although impacted by anthropogenic nutrient input has shown little or no measurable change in microbial productivity since added nutrients are rapidly transferred out of the photic zone via the by-pass and tunnelling processes and are exported from the basin. It is also suggested that fish productivity is higher than has been suggested by conventional food chain models due to this grazing. Two possible reasons for the unusual P-starved nature of the basin are presented. (c) 2005 Elsevier Ltd. All rights reserved.

Microbial uptake of orthophosphate was studied before and during a Lagrangian experiment where orthophosphate was added to the surface mixed layer in the Cyprus Gyre, Eastern Mediterranean, a region previously hypothesized to be characterized by P-limited growth of both phytoplankton and heterotrophic bacteria. The addition of ca. 110 nM orthophosphate to a ca. 16 km 2 patch in situ led, within 1 day, to an increase in particulate-P from 8 to ca. 15 nM, a result in good agreement with a previous microcosm bioassay indicating this system to have a maximum capacity for orthophosphate consumption of between 10 and 25 nM phosphate. In samples of unperturbed water taken before the addition, outside, or below the experimental patch, orthophosphate turnover time (T-t) was < 4 h, argued to be consistent with the assumption of diffusion-limited phytoplankton growth. Upon addition, Tt increased to 94h. Estimates of maximum potential uptake rate (V-max) for orthophosphate in unperturbed water exceeded by more than one order of magnitude the biological P-requirement (v) as obtained from stoichiometric conversion of C-based primary and bacterial production values to estimated P-requirement. Upon addition of orthophosphate, V-max decreased to a level comparable to v. The observations are consistent with the assumption of P-starved cells before and P-replete cells with excess external orthophosphate after the addition. Orthophosphate uptake in unperturbed water was dominated by < 1 pm organisms (mean + SD between samples 0.56 +/- 0.03 mu m). In samples with higher turnover time, orthophosphate uptake was shifted towards larger organisms, culminating after 5 days with a near doubling in mean size (1.08 mu m). The size distribution of particulate-P standing stock had a mean size of 10 pm, indicating the presence of a substantial biomass of micro-organisms larger than those involved in P-uptake. Comparison of the measured particulate-P with microscope- based biomass estimates indicated a microbial food web dominated by heterotrophic organisms (70% of particulate-P), distributed with ca. 25% of total particulate-P in heterotrophic bacteria, ca. 40% in heterotrophic flagellates, and ca. 5% in ciliates. Concentration of bioavailable phosphate (S-n) estimated from the relationship Sn = vTi indicated Sn values < 1 nM PO4 before the addition, increasing afterwards. Estimates of the sum Kt + Sn for the 0.6-0.2 put size fraction were in the range 1-7 nM PO4 before and outside patch, suggesting this sum to be dominated by the half-saturation constant K-t. K-t + S-n increased to 69 nM after addition, then dropped over the following week back to background levels. As reported elsewhere in this volume, there was a decline in the observed chlorophyll concentrations, but a positive response in copepods. Less clear than the effects at the level of osmotroph physiology were the subsequent responses expected in the food web. Two possible mechanisms are discussed: (1) a positive response in bacterial production and the subsequent food chain of bacterial predators, and (2) a positive response in phytoplankton predators due to a shift in food quality rather than in food quantity. (c) 2005 Elsevier Ltd. All rights reserved.

An on-board microcosm experiment was performed during the CYCLOPS May 2002 cruise to track the biogeochemical response of Eastern Mediterranean surface seawater to a gradient addition of fresh and pre-leached Saharan dust, mimicking the potential fertilization effect as opposed to the impact of adding particles alone. Response parameters examined were P-turnover time, bacterial production and abundance, chlorophyll a, other phytopigments, abundance of different pico and nanophytoplankton groups, primary production rates, abundance of heterotrophic nanoflagellates and ciliates. The addition of fresh Saharan dust (range: 0.2– 4.9 mg l - 1 ) and the subsequent nutrient release triggered an increase in phytopigments and primary production, while no response was detected for pre-leached dust particles. Most responses were linearly related to the amount of fresh dust added. Synechococcus and prymnesiophytes increased in abundance along with cellular pigment content while Prochlorococcus disappeared, heterotrophic bacteria increased production rates, and ciliates showed a small increase in cell density. A less clear response was recorded by in situ measurements following a Saharan dust storm during a cruise in the Levantine Basin in May 2001. The calculated amount of nutrients and dust particles delivered by such an event to a 15-m thick mixed surface layer is low ( ∼ 0.3 nmol P l - 1 , ∼ 9 nmol N l - 1 and 0.06 mg dust l - 1 ) , falling close to the lowest dust addition in our microcosm experiment. Even so, an enhancement of phosphate turnover time, a sharp decline of Prochlorococcus abundance, and slight increases in chlorophyll a and bacterial activity were observed in response to the dust storm. Considering the linear effect of fresh dust concentrations on the bacterial activity, primary production and pigment concentration (total and per cell), and the likely stimulation of grazing, it is not surprising that changes due to moderate strength dust storms are mostly close to detection limit of either field or remote sensing measurements.

An on-board microcosm experiment was set up to test the hypothesis that the observed lack of phytoplankton biomass increase response to a mesoscale in situ P-enrichment experiment in the P-limited Eastern Mediterranean (Krom et al., 2005a) was a consequence of co-limitation by P and N availability in this ultraoligotrophic environment. Six microcosms were filled with subsurface seawater (ambient DIN: 90-100 nM) taken from inside a P-enriched patch (IN), which in the absence of biological activity would have had ca. 22 nM of PO4-3. Another six microcosms were filled with unfertilized (< 2nM PO4-3) subsurface seawater from outside the patch (OUT). The bottles were either supplemented with 1600nM NH4 divided by or not, incubated on-deck and subsampled daily, or at the first and last (fourth) day of the experiment, for a suite of biological parameters. The addition of N to OUT water did not induce cell abundance increases in either the phototrophic or heterotrophic sides of the food chain, in line with previous assessments that the Eastern Mediterranean is not purely N-limited. The IN and OUT treatments, to which no NH4+ was added, mimicked the behavior of the in situ experiment, with an order of magnitude higher bacterial production of IN vs. OUT water, but no noticeable phytoplankton response. The addition of N to IN water, previously exposed to P, led to substantial responses of the entire microbial community, including 4 to 80-fold increases in chlorophyll, other pigments, bacterial activity, and the abundance of ciliates-relative to IN water to which N was not added. The ca. 10-fold increase in chlorophyll within 4 days was mostly due to a major increase in both abundance (x 4) and fluorescence per cell (x 17) of Synechococcus, whereas Prochlorococcus disappeared. These changes were accompanied by removal from the water of 570 nM of the added NH4+, equivalent to 570/22 or N:P ratio of 26:1, similar to the ratio measured for POM in the area. Possibly, non-Redfield ratios were maintained, still leaving by day 4 some 1100 nM of N that could not be used due to the lack of P. These results support our hypothesis that the lack of response of phytoplankton to the mesoscale P-enrichment was due to their concurrent N-starvation, i.e. N and P co-limitation. In contrast, bacteria could grow when only P was added, implying pure P-limitation. Thus, the heterotrophic and autotrophic components of the same aquatic community experienced different limitations. (c) 2005 Elsevier Ltd. All rights reserved.