**2.2. Exopolysaccharides of the biofilm matrix**

The ability to synthesize exopolysaccharides is widespread among microorganisms, and microbial exopolysaccharides play important roles in biofilm formation, pathogen persistence, and have several applications in the food and medical industries. Exopolysaccharides are considered to be important components of the biofilms matrix [27]. However, some studies suggest that exopolysaccharides may not always be essential for biofilm formation [28]. Most of the matrix exopolysaccharides are very long with a molecular weight of 500-2000 kDa. They can be homo-polymers such as cellulose, curdlan or dextran, or hetero-polymers like alginate, emulsan, gellan or xanthan. Exopolysaccharide chains can be linear or branched. They are generally constituted by monosaccharides and some non-carbohydrate substituents such as acetate, pyruvate, succinate, and phosphate [29]. Various examples of exopolysaccharides encountered in bacterial biofilm are presented in Table 2.

## **2.3. Carbohydrate content of exopolysaccharides**

Composition as well as conformation of sugar monomers may modify the properties of the exopolysaccharides and thus of the biofilm matrix. Mono-carbohydrate constituted exopolysaccharides are often D-glucose, D-galactose, D-mannose, L-fucose, L-rhamnose, Larabinose, N-acetyl- D-glucose amine and N-acetyl-D-galactose amine as well as the uronic acids D-glucuronic acid, D-galacturonic acid, D-manuronic acid and L-guluronic acid. Other sugar monomers less frequently occurring are D-ribose, D-xylose, 3-keto-deoxy-Dmannooctulosonic acid and several hexoseamineuronic acids [29]. Some examples of carbohydrate content in biofilm are presented in Table 3.

In conclusion of this section, it is clear that the extraction of exopolysaccharides from biofilms usually require a multi-method protocol. Furthermore, there is no standard extraction procedure established, making difficult the meaning, comparison and interpretation of published results. However, recent studies tend to evaluate whether molecular diversity of EPS are potential markers for biofilm macro-scale characteristics [40].



The content of the EPS extracts is done by chemical analyses. The exopolysaccharide content of EPS can be determined by the phenol-sulphuric acid method described by Dubois *et al.* [22] or by using the anthrone method according to Dreywood [23], with glucose as the standard. The protein content of EPS can be determined by the Bradford method [24] or by

As mentioned by several authors, yields of EPS extracted from biofilms depend on the extraction method used. Pan *et al.* [26] reported that chemical methods significantly increased the extraction yield from natural biofilm compared to physical methods. Nevertheless, the use of chemical methods can modify the composition of the EPS extracted. Indeed, added chemical extractants such as EDTA or formaldehyde can be present in the EPS extracts as contaminants and then can modify the EPS quantification efficiency. Moreover, chemicals can induce the release of intracellular components from treated cells and contaminate extracellular substances

The ability to synthesize exopolysaccharides is widespread among microorganisms, and microbial exopolysaccharides play important roles in biofilm formation, pathogen persistence, and have several applications in the food and medical industries. Exopolysaccharides are considered to be important components of the biofilms matrix [27]. However, some studies suggest that exopolysaccharides may not always be essential for biofilm formation [28]. Most of the matrix exopolysaccharides are very long with a molecular weight of 500-2000 kDa. They can be homo-polymers such as cellulose, curdlan or dextran, or hetero-polymers like alginate, emulsan, gellan or xanthan. Exopolysaccharide chains can be linear or branched. They are generally constituted by monosaccharides and some non-carbohydrate substituents such as acetate, pyruvate, succinate, and phosphate [29]. Various examples of exopolysaccharides

Composition as well as conformation of sugar monomers may modify the properties of the exopolysaccharides and thus of the biofilm matrix. Mono-carbohydrate constituted exopolysaccharides are often D-glucose, D-galactose, D-mannose, L-fucose, L-rhamnose, Larabinose, N-acetyl- D-glucose amine and N-acetyl-D-galactose amine as well as the uronic acids D-glucuronic acid, D-galacturonic acid, D-manuronic acid and L-guluronic acid. Other sugar monomers less frequently occurring are D-ribose, D-xylose, 3-keto-deoxy-Dmannooctulosonic acid and several hexoseamineuronic acids [29]. Some examples of

In conclusion of this section, it is clear that the extraction of exopolysaccharides from biofilms usually require a multi-method protocol. Furthermore, there is no standard extraction procedure established, making difficult the meaning, comparison and interpretation of published results. However, recent studies tend to evaluate whether molecular diversity of EPS are potential markers for biofilm macro-scale characteristics [40].

using the bicinchoninic acid reagent [20] with bovine serum albumin as the standard.

by intracellular material. These contaminants may be eliminated.

**2.2. Exopolysaccharides of the biofilm matrix** 

encountered in bacterial biofilm are presented in Table 2.

**2.3. Carbohydrate content of exopolysaccharides** 

carbohydrate content in biofilm are presented in Table 3.


(\*) not indicated

**Table 3.** Carbohydrate content of various biofilms
