Sampling, separation, and quantification of N-acyl homoserine lactones from marine intertidal sediments

N-acyl homoserine lactones (AHLs) are molecules produced by many Gram-negative bacteria as mediators of cell-cell signaling in a mechanism known as quorum sensing (QS). QS is widespread in marine bacteria regulating diverse processes, such as virulence or excretion of polymers that mediate biofilm formation. Associated eukaryotes, such as microalgae, respond to these cues as well, leading to an intricate signaling network. To date, only very few studies attempted to measure AHL concentrations in phototrophic microbial communities, which are hot spots for bacteria-bacteria as well as microalgae-bacteria interactions. AHL quantification in environmental samples is challenging and requires a robust and reproducible sampling strategy. However, knowing about AHL concentrations opens up multiple perspectives from answering fundamental ecological questions to deriving guidelines for manipulation and control of biofilms. Here, we present a method for sampling and AHL identification and quantification from marine intertidal sediments. The use of contact cores for sediment sampling ensures reproducible sample surface area and volume at each location. Flash-freezing of the samples with liquid nitrogen prevents enzymatic AHL degradation between sampling and extraction. After solvent extraction, samples were analyzed with an ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) method that allows to baseline-separate 16 different AHLs in less than 10 min. The sensitivity of the method is sufficient for detection and quantification of AHLs in environmental samples of less than 16 cm(3)

Methodology of light response curves: application of chlorophyll fluorescence to microphytobenthic biofilms

The light response curve methodology for microphytobenthic biofilms was studied by comparing the two most usual approaches used in pulse amplitude modulated (PAM) fluorometry. The non-sequential light curve (N-SLC) method is characterized by independent measures of the photosynthetic activity across a light gradient whereas the rapid light curve (RLC) method consists of successive measures on the same sample exposed to a stepwise increase of light intensities. Experiments were carried out on experimental microphytobenthic biofilms prepared from natural assemblages and acclimated to dark conditions. In preliminary experiments, N-SLCs were constructed from fluorescence induction curves performed at 12 different photon flux densities (PFDs). A minimum of 50 s of illumination was necessary to reach a stable light response curve; shorter illumination times resulted in underestimating the physiological parameters (α the light utilization coefficient in light-limited conditions and rETRmax the maximum rate of photosynthesis efficiency) of the light response curve. For the comparison between N-SLCs and RLCs, the same time of illumination (50 s) was used for each light step of RLCs so that N-SLCs differed from RLCs only by the way the amount of light was delivered, i.e., a light dose accumulation for RLC. The experimental results showed the difference between the two photobiological response curves. In the lower range of PFDs, RLCs exhibited a larger value of α; in this light-limited part of the response curve the incremental increase of PFDs limited the development of NPQ and resulted in a better optimization of electron transport rate for RLC. In the higher range of PFDs, the trend was reversed and the RLC showed a lower value of rETRmax than the N-SLC did; this is attributed to the light dose accumulation which likely led to a more efficient dispersion of energy, as illustrated by a higher non-photochemical quenching (NPQ). In conclusion, these results confirm that parameters derived from both methods differ in their value and do not bear the same physiological information.

Sensitivity evaluation of the green alga Chlamydomonas reinhardtii to uranium by pulse amplitude modulated (PAM) fluorometry

International audienceAlthough ecotoxicological studies tend to address the toxicity thresholds of uranium in freshwaters, there is a lack of information on the effects of the metal on physiological processes, particularly in aquatic plants. Knowing that uranium alters photosynthesis via impairment of the water photo-oxidation process, we determined whether pulse amplitude modulated (PAM) fluorometry was a relevant tool for assessing the impact of uranium on the green alga Chlamydomonas reinhardtii and investigated how and to what extent uranium hampered photosynthetic performance. Photosynthetic activity and quenching were assessed from fluorescence induction curves generated by PAM fluorometry, after 1 and 5h of uranium exposure in controlled conditions. The oxygen-evolving complex (OEC) of PSII was identified as the primary action site of uranium, through alteration of the water photo-oxidation process as revealed by F0/Fv. Limiting re-oxidation of the plastoquinone pool, uranium impaired the electron flux between the photosystems until almost complete inhibition of the PSII quantum efficiency (F'q/F'm, EC50=303±64μgUL-1 after 5h of exposure) was observed. Non-photochemical quenching (qN) was identified as the most sensitive fluorescence parameter (EC50=142±98μgUL-1 after 5h of exposure), indicating that light energy not used in photochemistry was dissipated in non-radiative processes. It was shown that parameters which stemmed from fluorescence induction kinetics are valuable indicators for evaluating the impact of uranium on PSII in green algae. PAM fluorometry provided a rapid and reasonably sensitive method for assessing stress response to uranium in microalgae. © 2013 Elsevier B.V

Does the size of the microphytobenthic biofilm on intertidal mudflats depend on the available photosynthetic biomass?

Many scientists consider that the top centimetre of the sediment on intertidal mudflats contains the photosynthetic competent biomass (PCB). Part of this biomass migrates upward to the surface of the sediment during diurnal emersion periods to form a temporary biofilm: the photosynthetic active biomass (PAB). The present study tests the hypothesis that the size of the biofilm (PAB) is functionally dependent on PCB. Therefore, we have plotted PAB as a function of PCB for a range of different environmental conditions: 3 seasons and 5 tidal situations, representing a total of 600 cores. This investigation points out that, in spring and autumn, there was a simple linear relationship between PAB and PCB, thus indicating that the maximum size of the biofilm represents a constant fraction (between 13 and 18 %) of the available biomass in the top centimetre of the mud. The relationship was more complex in winter, but the general trend remained the same. The dependence of the size of the biofilm on PCB might have important ecological implications in terms of primary productivity and also the monitoring of intertidal microalgal biomass at large spatial scales

Phototrophic Biofilm Activity and Dynamics of Diurnal Cd Cycling in a Freshwater Stream

Diel cycles of dissolved cationic metal concentrations commonly occur in freshwater streams in apparent response to coincident cycles in water quality parameters (pH, O2, temperature). Hourly sampling of the Cd-contaminated Riou Mort (France) revealed large diel cycles in “total” dissolved Cd (232−357 nM; <0.45 μm) and “truly” dissolved Cd (56−297 nM; <0.02 μm) which were strongly correlated with changes in water pH. Using measured fluxes, a dissolved O2 model was constructed that indicated that benthic metabolic activities, respiration and photosynthesis, were responsible for the diel O2 (and thus, CO2 and pH) variation in the stream. However, microsensor measurements also showed that the pH changes occurred at the biofilm interface earlier than in the bulk water column. This difference in timing was reflected in the Cd dynamics, where pH-controlled sorption effects caused Cd partitioning from the truly dissolved pool onto the biofilm in the morning, and from the truly dissolved pool onto large colloids (0.02−0.45 μm) later in the day. Because this process causes large changes in the bioavailable Cd fraction, it has significant implications for Cd toxicity in freshwater streams. This study demonstrates the profound control of benthic microbiological processes on the cycling of heavy metals in aquatic systems

Toxicité et bioaccumulation du cadmium dans les biofilms d'eau douce

[Departement_IRSTEA]MA [TR1_IRSTEA]QSA / DYNAQInternational audienceA microcosm study was undertaken to examine the effects of dissolved cadmium at various concentrations (0, 10, and 100 μg · L−1) on biofilm accumulation and diatom assemblages. A natural biofilm sampled from the Riou-Mort River (Southwest France) was inoculated into three experimental systems, where biofilm settled on glass slides. Samples collected after 1, 2, 4, and 6 weeks of colonization were analyzed for metal accumulation (total metal content and intracellular metal content in the biofilm), biomass (as measured through dry weight and ash-free dry matter), and quantitative as well as qualitative analysis of diatom assemblages. There was a positive correlation between cadmium accumulation and dissolved cadmium concentrations and duration of exposure: a linear relationship was found between concentration factors (CFs) of growing biofilms and time (CFs/day = 0.25 and 0.38 under contaminations of 10 and 100 μgCd · L−1, respectively). Biofilm settlement, more than photosynthetic activity, was affected by high cadmium concentrations: we observed for all stages of settlement a drastic and significant (p < 0.05) reduction in biofilm biomass and in diatom densities in the highest cadmium contamination, compared to control and low cadmium concentration units

Variations saisonnières de l'accumulation de cadmium par les communautés périphytiques en cours d'eau

[Departement_IRSTEA]MA [TR1_IRSTEA]QSA / DYNAQInternational audienceThe relationships between diatom species and cadmium (Cd) accumulated in biofilms of the Riou-Mort River (SW, France) were studied in July 2004 and March 2005. Biofilms were sampled from artificial substrates immersed along a metallic pollution gradient during 20 days. Dynamics of diatom communities and cadmium accumulation were followed by collecting samples after 4, 7, 14 and 20 days of biofilm colonization. Cd accumulation in biofilms during experiment was significantly higher in Cd polluted station (Joanis) than in reference station (Firmi) for both seasons. Periphytic diatom composition varied between sites and seasons. At Firmi station, seasonal dynamics of diatom communities were stable with the dominance of Cyclotella meneghiniana and Melosira varians in July and Surirellabrebissonnii and Navicula gregaria in March. At Joanis station, diatom communities mainly responded to high levels of metal by a high proportion of small, adnate species. Positive correlations between Eolimna minima, Nitzschia palea, Encyonema minutum, Surirella angusta, and Gomphonema parvulum and cadmium accumulation were observed, indicating that these species are tolerant to high levels of cadmium. On the other hand, negative correlations of C. meneghiniana, N. gregaria, Navicula lanceolata, M. varians and Nitzschia dissipata with cadmium qualify them as sensitive diatom species. Periphytic diatom composition through the presence of specific species highlight metal tolerant indicator diatom groups which will be meaningful for biomonitoring pollution in natural aquatic systems