# Static Magnetic Fields Effects on Polysaccharides Production by Different Microsalgae Strains
## 2 Materials and Methods
### Polysaccharides Extraction and Characterization
#### 2.3.2 EPS from Spirulina  
EPS from _Spirulina_ biomass were extracted according to [PERSON], et al. [44]. The biomass was resuspended in pH 9.5, 0.05 M TAPS ([tris (hydroxymethyl) methylamino] propanesulfonic acid) buffer containing 0.025 M EDTA and 0.025 M NaCl. The use of this buffer aims to limit cellular lysis during subsequent steps. After heating at 100 \({}^{\circ}\)C for 1 h, the suspension was centrifugated (17,000\(\times\)\(g\), 30 min, 20 \({}^{\circ}\)C) and the pellet was resuspended in the same buffer for a second extraction. The supernatants were combined, and acidic polysaccharides were precipitated with 10 volumes of 3% cetyltrimethylammonium bromide (CTAB) for 24 h. After centrifugation, the pellet was purified by 4 successive cycles of solubilization in KCl 1.5, 0.75 and 0.3 M, and a last resolubilization in milli-Q water. Between each solubilization in KCl, polysaccharides were precipitated with 3 volumes of 96\({}^{\circ}\) ethanol (left overnight at \(-\)20 \({}^{\circ}\)C) and the mixture was centrifugated to recover the polysaccharides pellet. These successive steps aimed to increase the purity of samples by removing most of the proteins and other impurities [45]. The samples were lyophilized for the next analyzes.  
Total carbohydrate content of EPS was analyzed by the resorcinol assay as described by [PERSON], et al. [46] using glucose as standard. Another standard curve was performed, using glucuronic acid, in order to access to the neutral sugar content using the corrective formula described by [PERSON], et al. [47]. Uronic acids were evaluated with meta  
Figure 2: Scheme of the photobioreactors used for _A. platensis_ SAG 2199 (**A**), _C. fusca_ and _Spirulina_ sp. (**B**) assays. 1 represents the ferrite magnets place in the photobioreactors; X represents the SMF measurements points.  
hydroxyldiphenyl, as described by [PERSON] and [PERSON] [48] using glucuronic acid as standard.  
Monosaccharide analysis was conducted as previously described [49; 50; 51; 52]. Acid hydrolysis were conducted with 2 M HCl for 90 min at 120 \({}^{\circ}\)C. Samples were neutralized with NH\({}_{4}\)OH 35%, centrifugated (15,000\(\times\)_g_, 15 min) and analyzed for their monosaccharide composition by High Pressure Anion Exchange Chromatography (HPAEC) equipped with pulsed amperometric detection (PAD). An ICS 3000 and AS 50 autosampler (Dionex, Sunnyvale, CA, USA) were used to inject 25 \(\mu\)L of samples on a guard CarboPac PA1-column (4 \(\times\) 50 mm) and analytical CarboPac PA1-column (4 \(\times\) 250 mm). Elution was performed using 18 mM NaOH for 25 min (to recover neutral sugars), followed by a linear gradient of 1 M sodium acetate in 200 mM NaOH to elute acidic monosaccharides. Data were collected and analyzed with Dionex Chromeleon 6.80 software (Sunnyvale, CA, USA). Quantification of monosaccharides was performed by plotting the response area as function of concentration of injected standards (L-Fuc, L-Ara, L-Rha, d-GlcN, d-Gal, D-Glc, D-Xyl, d-Man, d-Glc, d-GalA) in the range 0.001 and 0.05 g L\({}^{-1}\).