**2. Exopolysaccharides extraction and purification from the biofilm matrix**

This section focuses on specific extraction methods targeting exopolysaccharides. General extraction methods for exopolysaccharides are first presented, followed by a presentation of the corresponding exopolysaccharides properties and carbohydrate contents.

## **2.1. Methods for exopolysaccharides extraction**

Exopolysaccharides constitute the main EPS in many biofilms. They form the backbone of a network where other EPS components can be included. The stability of the biofilm matrix is dominated by entanglement of EPS and weak physicochemical interactions between molecules. These interactions correspond to various binding forces such as electrostatic attractive forces, repulsive forces (preventing collapsing), hydrogen bonds, van der Waals interactions and ionic attractive forces [13].

372 The Complex World of Polysaccharides

about 30% [12].

membranes used for drinking water production.

**2.1. Methods for exopolysaccharides extraction** 

heterologous ions and molecules to yield gels, often with multivalent cations playing a significant role in the process. Polysaccharides also interact with proteins molecules both as solutes and when attached to the surface of the microbial cells. The polysaccharide - protein interactions in the matrix induce both structural and functional properties. Indeed, some of

Biofilms in differing environments can be exposed to a very wide range of hydrodynamic conditions, which greatly affect the matrix and the biofilm structure [8]. The shear rate determines the rate of erosion of cells and regions of the matrix from the biofilm. Polysaccharides of the matrix exhibit flow and elastic recovery; because of the flexibility of the matrix its shape can change in response to an applied force. The shear stress to which a biofilm is exposed also affects the physical morphology and dynamic behaviour. Biofilms grown under higher shear are more strongly adhered and have a stronger EPS matrix than those grown under lower shear [9]. Biofilm density can be influenced by the fluid shear during growth [10]. *Pseudomonas* biofilms grown under laminar flow generally consist of hemispherical mound-shaped microcolonies, which form an isotropic pattern on the surface [9]. The biofilm microcolonies grown in turbulent flow are elongated in the downstream direction to form filamentous streamers. The streamers are attached to the glass substratum by an upstream head while the downstream tails are free to oscillate in the flow. Thus, hydrodynamics conditions influence both the structure and the material properties of biofilms [9]. This may be related to the physical arrangement of individual polymer strands in the biofilm EPS matrix [11]. The constitution of the biofilm matrix of *S. enteritidis* varies with pressure forces applied to the biofilm. Indeed in the absence of pressure, the sugars in the biofilm matrix are mainly composed of glucose and very little fucose. However in the presence of power flow, the share of fucose in the biofilm matrix is increased from 11% to

In this chapter, after the presentation of exopolysaccharides extraction and purification from the biofilm matrix, the structural and physical properties of bacterial alginates, cellulose and other exopoysaccharides related to biofilm formation are discussed. An illustration of the complexity of the biofilm matrix architecture and the role of exopolysaccharides in the properties of the matrix is given through biofilms formation at the surface of nanofiltration

**2. Exopolysaccharides extraction and purification from the biofilm matrix** 

This section focuses on specific extraction methods targeting exopolysaccharides. General extraction methods for exopolysaccharides are first presented, followed by a presentation of

Exopolysaccharides constitute the main EPS in many biofilms. They form the backbone of a network where other EPS components can be included. The stability of the biofilm matrix is

the corresponding exopolysaccharides properties and carbohydrate contents.

these proteins are enzymes constituting an external digestion system [7].

**Figure 1.** Schematic representation of a mature biofilm. In the centre, overall diagram of the structure of a biofilm to an interface solid / liquid: bacteria are attached to the solid surface and included in a selfinduced polymer matrix. In the area of contact between bacteria and surface, the microbial cells can interact with the surface via several protein and polysaccharide appendages (pili, flagella, LPS, capsular polysaccharides) depending on the type of bacteria. On the basis of the biofilm, bacterial cells are embedded in a matrix containing high eDNA concentrations, in addition to proteins and polysaccharides. The eDNA plays a major role in early biofilm formation. In the core of the biofilm, channels of water carrying ions and nutrients cross the biofilm matrix containing high concentrations of exopolymeric substances. All these exocellular compounds form a protective gel around the microorganisms. In the biofilm detachment area, microbial enzymes destroy the exopolymeric matrix and release the cells that regain mobility, to be able to colonize new surfaces.

The exopolysaccharides recovery from the biofilm matrix in order to get a better understanding of their nature, requires to break down the interactions between EPS and selectivity separate them from other EPS and from matrix cells without cell lysis. The

evaluation of cell lysis can be performed by measuring activity of the intracellular marker enzyme glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49). Thus, substantial cell lysis occurring during the EPS extraction is commonly observed [14]. Regarding extraction methods, publications dealing with selective extraction of exopolysaccharides are missing, as already reviewed by Denkaus *et al.* [15]. Indeed, procedures described in the literature mainly deals with EPS extraction.

Physical and/or chemical methods are used to extract EPS from biofilms. Some EPS are tightly associated to the biofilm structure, sometimes through covalent bounds to the cells surface and are not directly extracted. Others free EPS are directly released. The easily released EPS can be separated using physical methods such as high-speed centrifugation and ultrasonication. Indeed, centrifugation is often used to separate soluble EPS from bacterial cells from pure cultures. Firmly cells-associated EPS require chemical methods of extractions. EPS cross-linked by divalent cations can be released from the biofilm matrix by complexing agents such as ethylenediamine tetraacetic acid (EDTA), by cation-exchange resins such as Dowex or by a formaldehyde treatment with or without sodium hydroxide [14, 16].

Various methods used to extract EPS can be applied to the extraction of exopolysaccharides as illustrated on Figure 2.

**Figure 2.** Pathways of exopolysaccharides extraction methods from biofilms

EPS extraction can be done from pure cultures of from complex microbial communities. For example, EPS material can be removed from *Pseudomonas aeruginosa* bacteria by centrifugation at 40 000g for 2 hours at 10°C. Purification of alginate is mostly obtained by precipitation in the presence of organic solvents from culture supernatants and treatment with enzymes such as nucleases and proteases to remove contaminating nucleic acids and proteins [14]. EPS from activated sludge samples can also be extracted by a centrifugation protocol. Then, residual bacteria can be removed from the supernatant by filtration on cellulose acetate membranes (0.2 µm). For removal of low molecular weight material, the supernatants can be dialyzed against deionised water. Depending on the studies, dialysis tubings can have various molecular weight cut-off. Then, dialysate is concentrated by lyophilisation. Other processes of exopolysaccharides extraction from various biofilm species are presented in Table 1.

374 The Complex World of Polysaccharides

mainly deals with EPS extraction.

as illustrated on Figure 2.

evaluation of cell lysis can be performed by measuring activity of the intracellular marker enzyme glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49). Thus, substantial cell lysis occurring during the EPS extraction is commonly observed [14]. Regarding extraction methods, publications dealing with selective extraction of exopolysaccharides are missing, as already reviewed by Denkaus *et al.* [15]. Indeed, procedures described in the literature

Physical and/or chemical methods are used to extract EPS from biofilms. Some EPS are tightly associated to the biofilm structure, sometimes through covalent bounds to the cells surface and are not directly extracted. Others free EPS are directly released. The easily released EPS can be separated using physical methods such as high-speed centrifugation and ultrasonication. Indeed, centrifugation is often used to separate soluble EPS from bacterial cells from pure cultures. Firmly cells-associated EPS require chemical methods of extractions. EPS cross-linked by divalent cations can be released from the biofilm matrix by complexing agents such as ethylenediamine tetraacetic acid (EDTA), by cation-exchange resins such as Dowex or by a

Various methods used to extract EPS can be applied to the extraction of exopolysaccharides

**BACTERIAL BIOFILM MATRIX**

Chemical

use of buffer solution, EDTA, cation-exchange resin, formaldehyde, sodium hydroxide

EXOPOLYSACCHARIDES

Enzymatic

treatment with amylase, nuclease, protease

extraction

extraction

formaldehyde treatment with or without sodium hydroxide [14, 16].

**Figure 2.** Pathways of exopolysaccharides extraction methods from biofilms

Physical

centrifugation ultracentrifugation sonication filtration heating dialysis lyophilization

extraction


\*HPAEC-PAD: High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection

**Table 1.** Extraction condition and determination of exopolysaccharides from various biofilm species

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

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 by intracellular material. These contaminants may be eliminated.
