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The mechanism of iron uptake by the dry biomass of the brown seaweed Sargassum fluitans was investigated at the molecular level using different instrumental techniques. Transmission electron microscopy (TEM) and the chemical microanalysis by electron dispersive spectroscopy (EDS) of the biomass exposed to iron solutions confirmed the deposition of the metal mainly in the cell wall. The analysis of the Fe(II)-exposed biomass by X-ray photoelectron spectroscopy (XPS) indicated the presence of Fe in two states of oxidation in the biomass exposed to ferrous iron solution, whereas only Fe(III) was present in ferric ironexposed biomass, suggesting a partial oxidation of Fe(II) when in contact with the biomass. In both Fe(II)-and Fe(III)exposed samples, XPS indicated iron complexation with sulfate groups in the biomass. The FTIR analysis of metalloaded biomass samples revealed the chelating character of the ion complexation to carboxyl groups as well as the complexation of Fe(III) with sulfur of sulfonate groups in the biomass. This work confirmed the participation of carboxyl groups in the uptake of both Fe(II) and Fe(III), and of sulfonate groups in the uptake of Fe(III) by S. fluitans biomass.

The green fluorescent protein (GFP) was used as a model protein to study the recombinant protein production by the strain Methylobacterium extorquens ATCC 55366. Scale-up from shake flasks to 20 l fed-batch fermentation was achieved using methanol as a sole carbon and energy source and a completely minimal culture medium. Two different expression vectors were used to express GFP. Clone PCM-GFP containing the vector pCM110 with native promoter of the methanol dehydrogenase P mxaF produced approximately 100-fold more GFP than the clone PRK-GFP containing the vector pRK310 with the heterogeneous promoter P lac . Several fed-batch fermentations with and without selective pressure (tetracycline) were run in a 20 l stirred tank fermenter using the two different clones of M. extorquens. The methanol concentration was monitored with an on-line semiconductor gas sensor in the culture broth. It was maintained at a non-toxic level of 1.4 g l 31 with an adaptative control which regulates the methanol feed rate. The same growth profile was achieved in all fermentations. The maximum growth rate (W max ) was 0.18 h 31 with an overall yield (Y X =S ) of 0.3 g g 31 methanol. With this high cell density fermentation process, we obtained high levels (up to 4 g l 31 ) of GFP with the clone PCM-GFP. The maximum specific GFP production (Y GFP=X ) with this clone was 80 mg g 31 representing approximately 16% of the total cell protein. Additional feeding of pure oxygen to the fermenter permitted a longer phase of exponential growth but had no effect on the total yields of biomass and GFP. The specific GFP production of clone PCM-GFP remained unaffected in the presence or absence of selective pressure (tetracycline), within the initial 50 h of the fermentation culture. These results suggest that M. extorquens ATCC 55366 could be an interesting candidate for overexpression of recombinant proteins.