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Structure and role of biological communities involved in the transport and transformation of persistent pollutants at the marine air-water interface (AIRWIN)

Deliverables

A total of 90 bacterial strains were isolated from the sea surface microlayer and underlying waters. These species were identified by sequence analysis and the growth recovery of these isolates was investigated after exposure to solar radiation. Bacterioneuston and bacterioplankton isolates originating from two sites of north-western Mediterranean sea were exposed to 6 different exposure times to a solar simulator. After exposure, the growth of each isolate was monitored and different classes of resistance were determined according to the growth pattern. Resistance to solar radiation was equally distributed within bacterionesuton and bacterioplankton. Similarly, pigmented bacteria were not more resistant to solar radiation than non-pigmented bacteria. Nevertheless, large interspecific differences among the 90 marine isolates were observed. Pseudoalteromonas and Alteromas genera and isolates belonging to the Cytopha/flavobacterium/Bacteroides group appeared to be highly resistant. However and despite these variations, most of the isolates were resistant to sunlight exposure. Resistance to solar radiation appeared to be mainly mediated by DNA repair mechanisms suggesting that physiological traits such as pigmentation have a less important role. To our knowledge, this is the first report describing the resistance of bacterioneuston isolates to simulated solar radiation. Further studies on highly resistant isolates will be helpful to better investigate the molecular mechanisms involved in the resistance of marine bacteria to solar radiation.
Photochemical reactivity of the organic matter collected from the SML was compared to those collected from the SSW. No significant production of dissolved inorganic nutrients (NO(3), NH(4), PO(4)) was observed in both waters after their exposure to the solar radiation during one day. Photoremineralization rates measured by oxygen consumption showed a large range of responses between the experiments conducted (0.1 up to 1mmol O(2) m{-3}d{-1}), but in most cases DOM from SML showed higher photoremineralization rates compared to DOM from SSW. When bacteria were grown on this pretreated material we found no consistent trend in bacterial production or respiration. This suggests that the dissolved organic matter in both, the SML and SSW was of similar quality and susceptibility towards irradiation and that the production of more labile DOM was counterbalanced by the production of refractory material. The dataset on the photoremineralization experiments and the bacterial activity on pretreated DOM are synthesized in Deliverable 7 available at the AIRWIN web site. No significant differences in photoinhibition of bacterial and primary production were observed between SML and SSW samples after their exposure to surface solar radiation during 8h. When samples were kept in the dark during 16h after this exposure, and subsequently exposed once more to surface solar radiation for 8h, no photoinhibition of bacterial production was observed during the second day in both types of samples. However, SML samples show a much more higher potential of growth compared to SSW samples during this 32h period of incubation. Again, these original results should be published in the coming months at before summer 2004.
The surface film of the hydrosphere covers more than 70% of the world�s surface. The surface microlayer (SML) or �skin� of oceans is a sink for natural and anthropogenic materials originating from both the atmosphere and the water column. Organisms living in this SML are called �neuston� and our knowledge on the biology of the SML is still in its infancy. Research of the sea surface microlayer requires the use of appropriate sampling techniques and strategies and the question of what is the most suitable device has not yet been answered. In the present study, we have compared the efficiency of the Harvey glass plate and the Garrett metal screen to analyse of wide range of microbiological parameters from viruses to ciliates in SML samples collected at two coastal stations along the north Mediterranean coast. Two types of membranes (Teflon and polycarbonate) were also used to collect the bacterioneuston. For most biological parameters, the metal screen was the most appropriate technique providing higher concentrations in the SML compared to the glass plate and therefore, the highest enrichments compared to underlying waters. Phytoneustonic parameters were generally depleted compared to the underlying water when sampled by the glass plate. The metal screen is more efficient than the glass plate to collect larger neustonic organisms. We also provide clear evidence that membranes strongly overestimate the abundance of bacterioneuston due to selective adsorption. We conclude that the classic screen method is the most suitable technique allowing a short sampling time and the collection of large sample volumes when a large diversity of parameters are simultaneously analysed. The sea surface microlayer (SML) is also a poorly characterized marine compartment regarding the organic matter accumulation and the atmosphere ocean exchanges of persistent organic pollutants (POPs). A major constraint for these studies has been its reliable sampling. In the present work, over 30 SML samples from two contrasting sites in the North-Western Mediterranean -Barcelona (Spain) and Banyuls-sur-Mer (France)- were collected using four different sampling devices, namely, glass plate, metal screen, Harvey�s roller and a surface slick sampler, and compared with the corresponding underlying water (15 samples). Enrichment factors (EF) regarding different parameters (individual and total PCBs, PAHs, DOC, POC and SPM) were compared using statistical tests. From the results obtained it was concluded that the MS is the more advantageous system for sampling organic pollutants in terms of sampling efficiency under a variety of meteorological conditions. Obviously, the sampling for trace metals analysis is restricted to the glass plate device. Similar conclusions were drawn for particulate and dissolved fatty acids, as well as for the hydrophilic compounds (carbohydrates and amino-acids). These results have been included in several publications that have been submitted to international journals. The thinner the layer collected, the closer the biological and chemical composition of the sample to the original distribution in the SML will be. Under this point of view, the GP is preferable. However, the MS offers the smallest contact area between the water film to be sampled and the sampler, which probably results in the lowest contamination of the SML. Furthermore, the MS offers the advantage to fit better the size range of neustonic organisms since large cells cannot attach to the surface of the glass plates. Another advantage is that MS collects larger volumes in shorter periods of time (around 10 litres per hour and per screen), something essential when a lot of parameters are to be analysed that require large water volumes. In contrast, the GP has a much lower sampling capacity (1 liter per hour and per plate). The time of sampling is of a great importance since the longer the time of sampling, the highest is the effect of temporal and spatial variability. Thus, we recommend the use of MS when the aim is the comparison of different parameters. In all cases, control experiments should be performed to check for the potential bias that can be introduced by the sampler itself. There is clear evidence from this study that membranes have important drawbacks and should not be used for quantification purposes. Both hydrophobic and hydrophilic membranes overestimated the concentration of living organisms by selective surface adsorption. However, sampling with membranes remains of interest for rapid and easy collection of bacteria living in the surface microlayer. Finally, the depth at which UW samples are collected must be carefully determined depending on the question to be answered, as it was illustrated by the depth profiles of dissolved carbohydrates in the first meter below the surface. Furthermore, any comparison with other reports in the literature should be made not only in reference to the sampler but also to this depth.
The diversity of bacterioneuston, phytoneuston and zoonzeuston was investigated. We suggested that the terms "bacterioneuston" and "phytoneuston" to design the pelagic organisms, which have passively accumulated at the surface, is rather problematical. Most of these organisms enrich the SML due the physical process of flotation. This is why the diversity of these organisms is more or less similar to that of planktonic organisms. Although a few specific neustonic species may exist, they do not dominate bacterial communities in the SML. For zooneuston, we only focus on flagellates and ciliates that can be collected by the metal screen, although this sampling method has probably limitations for the collection of large ciliates. These organisms are those who participate to the microbial food webs. For these organisms, the conclusion was similar to that for bacteria and phytoplankton since SML species were not different from UW species. However, the concentration of flagellates was highly increased in the SML. Therefore, our findings are not in agreement with those reported in the year�s 80th and the SML is not dominated by neustonic species of bacteria and phytoplankton. In contrast, the accumulation of bacteria and phytoplankton is probably the result of the flotation process and these accumulated cells support an important activity of flagellates and ciliates. For bacteria and in addition to the discovery of new genera and species, a few isolates were specifically found in the surface microlayer suggesting �neustonic� properties. These species were affiliated to Arthrobacter agilis, Dietzia maris, Bacillus firmus, Marinobacter litoralis, Microbulbifer maritimus, Pseudoalteromonas agarivorans and Pseudoalteromonas atlantica.
In a tentative to compare the structure of the microbial food webs between the SML and SSW, we selected a total of 20 sampling dates from four sampling campaigns at Barcelona (n=10) and Banyuls (n=10) stations. Among the 16 biological parameters and 5 chemical parameters which were analysed, only 5 parameters showed significant differences in enrichment factors (EF) between the two sites (i.e. autrophic flagellates, ciliates, chl a, chl b, and chl c) with higher concentrations measured at Barcelona compared to Banyuls. For most of the parameters, the concentrations measured in the SML were highly correlated with those measured in the SSW, and no relationship appeared between the subsurface water concentration and the EF of each parameter, suggesting that the productivity of the system does not seem to play a role in the importance of the EF. The number of significant relationships between two parameters in SML (n=35) and SSW (n=32) was quite similar, with 15 relationships specific to SML and 11 specific to SSW. A higher ratio between autotrophic biomass and bacterial biomass was observed in SML compared to SSW. Autotrophic flagellates (AFLAG) could represent a significant part of the phytoneuston with regard to their rapid colonization of the SML during periods of calm sea and the high correlation observed between the EF of AFLAG and the EF of chlorophyll a. To our knowledge this is the first study allowing the comparison of all the components of the microbial food web between SML and SSW.
The Sea Surface Microlayer (SML) and Underlying Water (UW) were investigated to assess the potential specificity of their biochemical structure in terms of lipid, carbohydrate and amino acid compositions. Particulate lipids, unlike what is usually found in the water column, were found to be more abundant than the dissolved moieties, and to be enriched in the SML whereas the dissolved species were equally partitioned between SML and UW. Dissolved hydrophilic molecules (combined and free amino acids and carbohydrates) were found, unexpectedly, more enriched in SML (both with a mean Enrichment Factor of 1.2) than dissolved lipids, with higher proportions of hydrophobic amino acids (isoleucine, alanine and glycine) in SML. Data recorded clearly indicate that the chemical and physical properties of the molecules are not the only ones to be involved in the processes contributing to the SML enrichment. At least three contributively sources to SML enrichment were inferred from the temporal variations of several specific biochemical markers, followed over a diel cycle at the Banyuls site: - Inputs of fresh phytoplanktonic matter from a pulse (6 hours) of C14-C22 fatty acids present in the triglycerides associated with large cells (>0.7 µm), - Inputs of zooplankton fecal pellets from a pulsed of chitine (polymerised glucosamine) increase, - And of bacterial populations from b-alanine (a bacterial metabolite) increase. The uppermost layer of the water column was shown to be an active source of organic matter export: fluxes of particles recorded at 40cm depth (at the Banyuls site) were mostly comprised of fresh organic matter (C14-C22 fatty acids) but also of terrigeneous matter (C24-C32 fatty acids) and of partly degraded lipids (Coloured Dissolved Organic Matter). In addition, higher concentrations of particulate and dissolved lipids and of dissolved free and combined amino acids were found at the Barcelona site, consistent with the more eutrophic levels of the ecosystem there than at Banyuls. An interannual variability exists in the levels of the biochemical species, as exemplified by the concentrations of these biochemical parameters that were higher in 2001 than in 2002.

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