Assessing the bioavailability of black carbon-derived dissolved organic matter for marine heterotrophic prokaryotes.

Here we investigated the bioavailability of black carbon (BC)-derived dissolved organic matter (DOM) for a natural mixed community of marine heterotrophic prokaryotes. We ran an in vitro biodegradation experiment that took place over 3 months and exposed a community of organisms collected in the northwestern Mediterranean Sea (Bay of Marseille, France) to three different soluble fractions of BC prepared in the laboratory from various fossil fuel combustion particulates: standard diesel (DREF), oxidized diesel (DREF-OX), and natural samples of ship soot (DSHIP). Over the course of the three months, we observed significant decreases in the concentrations of dissolved organic carbon (DOC; from 9 to 21 %), dissolved BC (DBC; from 22 to 38 %) and dissolved polycyclic aromatic hydrocarbons (d-PAH; from 24 to 64 %) along with variability in the growth dynamics and activity of the heterotrophic prokaryotic community. The heterotrophic prokaryotic community exposed to DREF-OX treatment showed the highest values of respiration and production and the highest cell abundance, associated with the highest decrease in DOC (21 %) and d-PAH (64 %) concentrations. In the DREF and DSHIP treatments, prokaryotic activity was oriented towards anabolism. DREF treatment led to the highest decrease in DBC concentration (38 %). DSHIP treatment, which presented a substantially different d-PAH and dissolved metals content to the other two treatments, showed the lowest decreases in DOC, DBC and d-PAH concentrations, as well as the lowest prokaryotic activity and biomasses. Our results indicate that BC-derived DOM, including the most condensed fraction of this material, is partly bioavailable and therefore likely to be assimilated by marine prokaryotes. The origin of BC/soot deposited at the ocean surface turns out to be a key parameter that dictates the efficiency of biodegradation of its dissolved fraction by heterotrophic prokaryotes.

Anomalously high abundance of Crocosphaera in the South Pacific Gyre.

The unicellular diazotrophic cyanobacterium Crocosphaera contributes significantly to fixed nitrogen inputs in the oligotrophic ocean. In the western tropical South Pacific Ocean (WTSP), these diazotrophs abound thanks to the phosphorus-rich waters provided by the South Equatorial Current, and iron provided aeolian and subsurface volcanic activity. East of the WTSP, the South Pacific Gyre (SPG) harbors the most oligotrophic and transparent waters of the world's oceans, where only heterotrophic diazotrophs have been reported before. Here, in the SPG, we detected unexpected accumulation of Crocosphaera at 50 m with peak abundances of 5.26 × 105 nifH gene copies l-1. The abundance of Crocosphaera at 50 m was in the same order of magnitude as those detected westwards in the WTSP and represented 100% of volumetric N2 fixation rates. This accumulation at 50 m was likely due to a deeper penetration of UV light in the clear waters of the SPG being detrimental for Crocosphaera growth and N2 fixation activity. Nutrient and trace metal addition experiments did not induce any significant changes in N2 fixation or Crocosphaera abundance, indicating that this population was not limited by the resources tested and could develop in high numbers despite the oligotrophic conditions. Our findings indicate that the distribution of Crocosphaera can extend into subtropical gyres and further understanding of their controlling factors is needed.

Dissolved organic matter stimulates N2 fixation and nifH gene expression in Trichodesmium.

Mixotrophy, the combination of heterotrophic and autotrophic nutrition modes, is emerging as the rule rather than the exception in marine photosynthetic plankton. Trichodesmium, a prominent diazotroph ubiquitous in the (sub)tropical oceans, is generally considered to obtain energy via autotrophy. While the ability of Trichodesmium to use dissolved organic phosphorus when deprived of inorganic phosphorus sources is well known, the extent to which this important cyanobacterium may benefit from other dissolved organic matter (DOM) resources is unknown. Here we provide evidence of carbon-, nitrogen- and phosphorus-rich DOM molecules enhancing N2 fixation rates and nifH gene expression in natural Trichodesmium colonies collected at two stations in the western tropical South Pacific. Sampling at a third station located in the oligotrophic South Pacific Gyre revealed no Trichodesmium but showed presence of UCYN-B, although no nifH expression was detected. Our results suggest that Trichodesmium behaves mixotrophically in response to certain environmental conditions, providing them with metabolic plasticity and adding up to the view that mixotrophy is widespread among marine microbes.

Mixotrophic metabolism by natural communities of unicellular cyanobacteria in the western tropical South Pacific Ocean.

Cyanobacteria are major contributors to ocean biogeochemical cycling. However, mixotrophic metabolism and the relative importance of inorganic and organic carbon assimilation within the most abundant cyanobacteria are still poorly understood. We explore the ability of Prochlorococcus and Synechococcus to assimilate organic molecules with variable C:N:P composition and its modulation by light availability and photosynthetic impairment. We used a combination of radiolabelled molecules incubations with flow cytometry cell sorting to separate picoplankton groups from the western tropical South Pacific Ocean. Prochlorococcus and Synechococcus assimilated glucose, leucine and ATP at all stations, but cell-specific assimilation rates of N and P containing molecules were significantly higher than glucose. Incubations in the dark or with an inhibitor of photosystem II resulted in reduced assimilation rates. Light-enhanced cell-specific glucose uptake was generally higher for cyanobacteria (∼50%) than for the low nucleic acid fraction of bacterioplankton (LNA, ∼35%). Our results confirm previous findings, based mainly on cultures and genomic potentials, showing that Prochlorococcus and Synechococcus have a flexible mixotrophic metabolism, but demonstrate that natural populations remain primarily photoautotrophs. Our findings indicate that mixotrophy by marine cyanobacteria is more likely to be an adaptation to low inorganic nutrient availability rather than a facultative pathway for carbon acquisition.

Determination of lipid degradation by marine lipase-producing bacteria: critical evaluation of lipase activity assays.

With the aim of obtaining a better understanding of lipids-lipases interactions in bacterioplankton communities in oceans, we used different methods for measuring lipase activities in pure cultures of the marine strain Alteromonas macleodii. The decay of tripalmitate added to cultures was followed chemically over time. In an enzymatic approach, lipase activities were measured using the fluorogenic lipid analogs MUF-palmitate and ELF-palmitate. When hydrolyzed by lipase, the non-fluorescent substrates release MUF and ELF Alcohol (ELFA) which are fluorescent. As shown by spectrofluorometry, ELF-palmitate was an efficient competitor for MUF-palmitate. However, the activities reached using these two fluorogenic substrates were different, but still much higher than the tripalmitate hydrolysis rate, measured chemically. MUF- and ELF-palmitate would not be hydrolyzed by lipase sensu stricto (defined as triacylglycerol acylesterase E.C. 3.1.1.3) but rather reflects lipolytic activities in a broad sense. ELFA is also water-insoluble and theoretically precipitates in the external membrane of bacteria causing its hydrolysis, which would allow microscopic identification of active cells. By epifluorescence microscopy, the accumulation of ELFA fluorescence over time was detected (as large, diffuse halos), but no precipitates were clearly associated with bacteria on slide preparations, neither for pure cultures of Alteromonas macleodii nor for natural samples from the Bay of Marseille, France. Among possible biases, those related to the hydrophobic/hydrophilic conditions required for precipitation are discussed.

Detection of extracellular phosphatase activity at the single-cell level by enzyme-labeled fluorescence and flow cytometry: the importance of time kinetics in ELFA labeling.

ELF97 phosphate (ELF-P) is a useful compound for assessing the phosphorus-related status of planktonic aquatic populations. The technique has been successfully applied to phytoplankton and more recently to heterotrophic prokaryotes in both freshwater and marine samples. We have used a recently developed protocol that enables the detection by flow cytometry of ELF alcohol (ELFA), the product of ELF-P hydrolysis. This protocol allows for identification of the fraction of cells able to express phosphatase activity (i.e., ELFA-labeled). This protocol is also very valuable in the study of time kinetics in this ELFA-labeling. The percentage of ELFA-labeled cells, the relative median ELFA fluorescence per cell, and the absolute ELFA fluorescence were determined in both freshwater (lake) and marine samples. The incubation time necessary to reach a stable percentage of active cells with maximal fluorescence intensity varied widely among samples. We highlight very subtle but important problems of discrimination between active and nonactive cells and of estimation of per-cell activity and we underline the importance of studying time kinetics of ELFA-labeling to determine the appropriate incubation time and thus making sample comparisons more relevant. Working on time kinetics of ELFA-labeling is promising for phosphomonoester hydrolysis rate determination at single cell level.

A method for analysing phosphatase activity in aquatic bacteria at the single cell level using flow cytometry.

It has been demonstrated that ELF97-phosphate (ELF-P) is a useful tool to detect and quantify phosphatase activity of phytoplankton populations at a single cell level. Recently, it has been successfully applied to marine heterotrophic bacteria in culture samples, the cells exhibiting phosphatase activity being detected using epifluorescence microscopy. Here, we describe a new protocol that enables the detection of ELF alcohol (ELFA), the product of ELF-P hydrolysis, allowing the detection of phosphatase positive bacteria, using flow cytometry. Bacteria from natural samples must be disaggregated and, in oligotrophic waters, concentrated before they can be analyzed by flow cytometry. The best efficiency for disaggregating/separating bacterial cell clumps was obtained by incubating the sample for 30 min with Tween 80 (10 mg l(-1), final concentration). A centrifugation step (20,000 g; 30 min) was required in order to recover all the cells in the pellet (only 7+/-2% of the cells were recovered from the supernatant). The cells and the ELFA precipitates were resistant to these treatments. ELFA-labelled samples were stored in liquid nitrogen for up to four months before counting without any significant loss in total or ELFA-labelled bacterial cell abundance or in the ELFA fluorescence intensity. We describe a new flow cytometry protocol for detecting and discriminating the signals from both ELFA and different counterstains (4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)) necessary to distinguish between ELFA-labelled and non ELFA-labelled heterotrophic bacteria. The method has been successfully applied in both freshwater and marine samples. This method promises to improve our understanding of the physiological response of heterotrophic bacteria to P limitation.

Evaluation of double formalin--Lugol's fixation in assessing number and biomass of ciliates: an example of estimations at mesoscale in NE Atlantic.

Ciliated protozoa are potential grazers of primary and bacterial production and act as intermediaries between picoplankton and copepods and other large suspension feeders. Accurate determination of ciliate abundance and feeding mode is crucial in oceanic carbon budget estimations. However, the impact of different fixatives on the abundance and cell volume of ciliates has been investigated in only a few studies using either laboratory cultures or natural populations. Lugol's solution and formalin are the most commonly used fixatives for the preservation of ciliates samples. In the present study, the aim was to compare 0.4% Lugol's solution and 2% borated-formalin fixation and evaluate the need of counting duplicate samples each using a different fixative. For this, a large number of samples (n = 110) from the NE Atlantic was analyzed in the frame of POMME program (Multidisciplinary Mesoscale Ocean Program). We established a statistically significant relationship (p < 0.0001) between Lugol's and formalin fixed samples for both abundance (r2 = 0.50) and biomass (r2 = 0.76) of aloricate ciliates which showed that counts were higher in Lugol's solution by a factor of 2 and a non-taxon specific cell-loss in formalin. However, loricate ciliate abundance in our samples which were represented primarily by Tintinnus spp. did not show any difference between the two treatments. Abundance and biomass of mixotrophic ciliates (chloroplast-bearing cells) were for various reasons underestimated in both treatments. Our results show that unique fixation by formalin may severely underestimate ciliates abundance and biomass although their population may not alter. For this reason, Lugol's solution is best for the estimation of their abundance and biomass. However, for counts of mixotrophs and the evaluation of the ecological role of ciliates in carbon flux, double fixation is essential. Compromises regarding the fixatives have lead to severe underestimations of mixotrophs in studies conducted by now.