## 3 Results
### Biofilm Water-Soluble Components
#### 3.4.4 Size Exclusion Chromatographic Profiles  
Each extract was analyzed using HPSEC-MALS to determine the molecular weight pattern. Elution profiles with refractive index (RI) and ultraviolet (UV) detection at 280 nm were obtained (Figure 8). The refractometric index is related to the amount of carbohydrates and nucleic acids, and UV profiles are related to proteins, peptides, and other components that absorb wavelength of 280 nm. Both chromatographic profiles were in accordance with the previous observations. Indeed, a large salt peak was detected using RI at the column total volume (12-min elution). This might partly explain the low level of the total amount of analyzed molecules (Figures 5 and 7). All extracts of _V. diabolicus_ exhibited three families of peaks (7-8 min, 8-10 min, and 10-11 min retention time) in the RI profile, corresponding to high-, medium-, and low-molecular-weight carbohydrates as determined by the MALS detector. Only SnP (planktonic cells supernatant) contained a high molecular weight carbohydrate with a small UV absorption. This could be attributed to an anionic polysaccharide. Medium-molecular-weight compounds were found in AB and AP extracts (NaOH extracts) with a low UV absorption. Both extracts presented quite similar peaks with a higher intensity for AP. RI profiles of MS969 extracts were quite different in molecular weight repartition. There was no very high molecular weight compound in SnB extract, but all the extracts featured a peak at 8 min elution time, which absorbed UV. The AP extract presented two peaks, at 8 and 9.5 min, whereas AB extract peaks were detected at 8 min and 10.5 min. The UV profiles were very similar from one extract to another. Figure 7: Osidic composition (% _w/w_) of _V. diabolicus_ and MS969 extracts. GalNAc: N-Acetylgalactosamine, GlcNAc: N-Acetylglucosamine, GalA: Galacturonic acid, Glc: Glucuronic acid, Glc: Glucose, Gal: Galactose, Man: Mannose, Fuc: Fucose, Rha: Rhamnose. Added inserts zoom on extracts containing low level of sugars. The osidic composition of EPS produced in the presence of 30 g/L glucose is indicated for both strains [54]. Since both _Vibrio_ strains produced EPSs of biotechnological importance when glucose was added to planktonic growth medium, we were interested in the identification of those produced by the biofilm and planktonic cells. [PERSON] et al. [15] suggested that EPSs present in biofilms certainly closely resemble the corresponding polymers synthesized by planktonic cells. The HE800 EPS is produced by _V. diabolictus_ in shaken flasks or in aerated bioreactors containing the Zobell medium supplemented with glucose; its linear tetrasaccharide repeating unit consists of two GlcA residues and two N-acetyl hexosamines: one GlcNAc and one GalNAc[68]. Although GC analysis of SnB gave only very low amounts of sugars, its overall oxidic profile, together with SnP, seemed to be similar to the HE800 EPS. However, mannose was present in these two extracts, suggesting that another compound was co-extracted. Sugars recovered in the AB extract were also in very low amounts but only glucose was identified. This may suggest that the glycopymer (as indicated by HPSEC-MALS) was similar to cellulose. Bacterial cellulose has already been described in biofilms of other Vibrionaceae, such as _Aliivibrio fischeri_[21]. Comparative sequence analyses also revealed the presence of a cellulose biosynthesis cluster in diverse bacteria, especially several Proteobacteria strains, of which the _Vibrio_ species is a member [21]. The EPSs produced in excess of carbon source (glucose) by the MS969 strain cultured in shaken flasks was composed of neutral sugars including rhamn, galactose, glucose, and glucuronic acid. Interestingly, it was the first time that a sulfate group, which is known to promote biological activities, was identified in an EPS produced by a _Vibrio_ strain [54]. The pink color seen in the electrophoresis in this study might also suggest that extracts prepared from MS969 (SnB, SnP, AB, and AP) contained sulfated compounds.