2D Image Quantification of Microbial Iron Chelators (Siderophores) Using Diffusive Equilibrium in Thin Films Method.

Siderophores are natural metal chelating agents that strongly control the biogeochemical metal cycles such as Fe in the environment. This article describes a new methodology to detect and quantify at the micromolar concentration the spatial distribution at millimeter scale of siderophores within the root's system. The "universal" CAS assay originally designed for bacterial siderophores detection and later designed for fungus was adapted here for diffusive equilibrium in thin film gel techniques (DET). The method was calibrated against the marketed desferrioxamine mesylate (DFOM) siderophore and applied with experiments performed with sunflower ( Helianthus annuus) and wheat ( Triticum aestivum) cultivated on free iron agar medium plates. We present here the first results with 2D images of the siderophores distribution in the vicinity of the root system of plants. With this technique we detected (i) the production of siderophores on bacteria inoculated ( Pseudomonas fluorescens) environments and (ii) hotspots of natural iron binding ligands production up to 50 µM in the wheat rhizosphere. The lower detection limit in our experiment was 2.5 µmol/L. This new technique offers a unique opportunity to investigate the siderophore production in two dimensions in a wide range of applications from laboratory experiments to natural systems very likely using an in situ and nondestructive tool.

Photosynthetic epibionts and endobionts of Pacific oyster shells from oyster reefs in rocky versus mudflat shores.

The Pacific oyster, Crassostrea gigas (Thunberg), is the main bivalve species cultivated in the world. With global warming enabling its reproduction and larval survival at higher latitudes, this species is now recognized as invasive and creates wild oyster reefs globally. In this study, the spatial distribution of photosynthetic assemblages colonizing the shells of wild C. gigas was investigated on both a large scale (two contrasting types of reefs found in mudflats and rocky areas) and a small scale (within individual shells) using a hyperspectral imager. The microspatial distribution of all phototrophs was obtained by mapping the Normalized Difference Vegetation Index (NDVI). Second derivative (δδ) analyses of hyperspectral images at 462, 524, 571 and 647 nm were subsequently applied to map diatoms, cyanobacteria, rhodophytes and chlorophytes, respectively. A concomitant pigment analysis was carried out by high performance liquid chromatography and completed by taxonomic observations. This study showed that there was high microalgal diversity associated with wild oyster shells and that there were differences in the structure of the phototropic assemblages depending on the type of reef. Namely, vertically-growing oysters in mudflat areas had a higher biomass of epizoic diatoms (hyperspectral proxy at δδ462 nm) and were mainly colonized by species of the genera Navicula, Nitzschia and Hippodonta, which are epipelic or motile epipsammic. The assemblages on the horizontal oysters contained more tychoplanktonic diatoms (e.g. Thalassiosira pseudonana, T. proschkinae and Plagiogrammopsis vanheurckii). Three species of boring cyanobacteria were observed for both types of reef: Mastigocoleus testarum, Leptolyngbya terrebrans, and Hyella caespistosa, but the second derivative analysis at 524 nm showed a significantly higher biomass for the horizontally-growing oysters. There was no biomass difference for the boring chlorophyte assemblages (δδ647 nm), with two species: Eugomontia testarum and Ostreobium quekettii observed for both types of reef. This study shows that oyster shells are an idiosyncratic but ubiquitous habitat for phototrophic assemblages. The contribution of these assemblages in terms of biomass and production to the functioning of coastal areas, and particularly to shellfish ecosystems, remains to be evaluated.

Simultaneous 2D imaging of dissolved iron and reactive phosphorus in sediment porewaters by thin-film and hyperspectral methods.

This study presents a new approach combining diffusive equilibrium in thin-film (DET) and spectrophotometric methods to determine the spatial variability of dissolved iron and dissolved reactive phosphorus (DRP) with a single gel probe. Its originality is (1) to postpone up to three months the colorimetric reaction of DET by freezing and (2) to measure simultaneously dissolved iron and DRP by hyperspectral imaging at a submillimeter resolution. After a few minutes at room temperature, the thawed gel is sandwiched between two monospecific reagent DET gels, leading to magenta and blue coloration for iron and phosphate, respectively. Spatial distribution of the resulting colors is obtained using a hyperspectral camera. Reflectance spectra analysis enables deconvolution of specific colorations by the unmixing method applied to the logarithmic reflectance, leading to an accurate quantification of iron and DRP. This method was applied in the Arcachon lagoon (France) on muddy sediments colonized by eelgrass (Zostera noltei) meadows. The 2D gel probes highlighted microstructures in the spatial distribution of dissolved iron and phosphorus, which are most likely associated with the occurrence of benthic fauna burrows and seagrass roots.

In vivo estimation of pigment composition and optical absorption cross-section by spectroradiometry in four aquatic photosynthetic micro-organisms.

The objective of the present study was to estimate in vivo pigment composition and to retrieve absorption cross-section values, a(∗), of photosynthetic micro-organisms using a non-invasive technique of reflectance spectrometry. To test the methodology, organisms from different taxonomical groups and different pigment composition were used (Spirulina platensis a Cyanophyta, Porphyridium cruentum a Rhodophyta, Dunaliella tertiolecta a Chlorophyta and Entomoneis paludosa a Bacillariophyta) and photoacclimated to two different irradiance levels: 25 µmol photonm(-2)s(-1) (Low Light, LL) and 500 µmol photonm(-2)s(-1) (High Light, HL). Second derivative spectra from reflectance were used to identify pigment in vivo absorption bands that were linked to specific pigments detected by high performance liquid chromatography. Whereas some absorption bands such as those induced by Chlorophyll (Chl) a (416, 440, 625 and around 675 nm) were ubiquous, others were taxonomically specific (e.g. 636 nm for Chl c in E. paludosa) and/or photo-physiological dependent (e.g. 489 nm for zeaxanthin in the HL-acclimated S. platensis). The optical absorption cross-section, a(∗), was retrieved from reflectance data using a radiative transfer model previously developed for microphytobenthos. Despite the cellular Chl a decrease observed from LL to HL (up to 88% for S. platensis), the a(∗) increased, except for P. cruentum. This was attributed to a 'package effect' and to a greater absorption by photoprotective carotenoids that did not contribute to the energy transfer to the core Chl a.

[Microphytobenthos assemblage mapping by spatial visible-infrared remote sensing in a shellfish ecosystem]. / Cartographie des peuplements du microphytobenthos par télédétection spatiale visible-infrarouge dans un écosystème conchylicole.

The aim of this work is to assess the use of (SPOT) multispectral visible infrared remote sensing to study microphytobentos assemblages in a shellfish ecosystem (Bay of Bourgneuf, France). SPOT satellite images (acquired at low tide in spring or autumn between 1986 and 1998) were calibrated using in situ radiometric data, and the normalised vegetation index (NDVI) obtained from these images showed microphytobenthos on bay mudflats. Proliferation was mainly along a north-south strip, essentially localised around the +2 m isobath and covering a surface area of 19 to 25% of the total mudflat area studied (420 to 550 ha). Three factors seem to be responsible for the spatial structure of the assemblages: bathymetry, nutrient input from the Falleron River and its channel, and the location of oyster-farming areas. Although spatial and spectral resolutions of multispectral remote sensing data have certain limitations, this approach opens up a new field of application for hyperspectral remote sensing, particularly for synoptic mapping of biomass distribution.