###### Abstract  
In the marine environment, biofilm formation is an important lifestyle for microorganisms. A biofilm is comprised of cells embedded in an extracellular matrix that holds them close together and keeps the biofilm attached to the colonized surface. This predominant lifestyle and its main regulation pathway, namely quorum-sensing (QS), have been shown to induce specific bioactive metabolites. In this study, we investigated the biofilm formation by two marine bacteria belonging to the _Vibrio_ species to discover potentially innovative bioactive compounds. We proposed a protocol to isolate biofilm extracts, to analyze their biochemical composition, and to compare them to planktonic cell extracts. Cells were grown attached to a plastic surface; extracts were prepared in water, NaOH, or in ethyl acetate and analyzed. Extracellular matrix components featured carbohydrates, proteins, lipids, and low amount of DNA. Carbohydrates appeared to be the main constituent of biofilm but also of the planktonic cell supernatant. Moreover, antimicrobial and QS-signaling activities were evidenced in extracts. _Vibrio_; biofilm; polysaccharide; quorum-sensing; activity +
Footnote †: journal: Cell  
is mainly produced by the cells themselves and can account for over 90% of the total dry mass of the biofilm [3]. A biofilm can be the cause of the corrosion and biofouling of marine equipment, resulting in its degradation and economic losses, and until now, most of the research efforts were focused on its inhibition [4]. In contrast, little is known about biofilm-specific metabolites. Indeed, a biofilm enables cell-cell interaction and communication by involving chemical signals, such as quorum-sensing (QS) autoinducers. Different metabolic pathways that remain inactive under the planktonic growth conditions are commonly used in the research laboratories where they can be activated [5; 6]. Many biosynthetic pathways are regulated by QS, including the production of metabolites [7; 8], of biosurfactants [9; 10], and of antimicrobials [11]. Biofilm-specific EPM substances may be endowed with diverse physical/chemical properties and with biological activities and functionalities. Polysaccharides (PSs) have been evidenced as the most common components of a bacterial biofilm matrix [12; 13; 14]. Specific PSs can be produced during biofilm formation and development [14; 15; 16]. For example, cellulose production has been described at the air-liquid interface of static liquid cultures of bacteria [6; 17]. Biotechnological applications of carbohydrate polymers are strongly influenced by their structural features and they have been investigated in various fields, including human health and the production of valuable metabolites [18; 19; 20; 21]. Proteins in biofilms are the structural components of multimeric cellular appendages, such as flagella, pili, or fimbiae. They are involved in cell motility and in both the initial phase of biofilm formation and in its development [17]. Some of them are located on the cell surface to promote interactions with other extracellular matrix ligands, such as LecB in _Pseudomonas aeruginosa_[22] or the biofilm-associated proteins (Bap) [17; 23]. Furthermore, many proteins are extracellular enzymes, such as proteases [12; 24], which could be worth studying for their biotechnological potential. Among other less characterized biofilm components, extracellular DNA (eDNA) has also been found in EPMs and may play a structural role in biofilm development by promoting cell-to-cell interconnections [25; 26; 27]. Extracellular DNA release also takes part in both genetic material exchange and gene transfer [28]. Data on lipids as EPM components are still limited. In biofilms, two main groups have been identified, namely lipid vesicles, in particular in _Vibrio fischeri_ biofilm [29], and lipoconjugates (glycolipid surfactants, lipopolysaccharide, lipoproteins) [30; 31; 32; 33]. It appears then that, while EPM substances act to provide structure for the biofilm, their biotechnological potential is broad.