Revised paradigm of aquatic biofilm formation facilitated by microgel transparent exopolymer particles Edo Bar-Zeeva, Ilana Berman-Franka, Olga Girshevitzb, and Tom Bermanc,1 aMina and Everard Goodman Faculty of Life Sciences and bCenter for Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan 52900, Israel; and cYigal Allon Kinneret Limnological Laboratory, Israel Oceanographic and Limnological Research, Migdal 14102, Israel Edited by Tom M. Fenchel, University of Copenhagen, Helsingor, Denmark, and approved April 30, 2012 (received for review March 2, 2012) Transparent exopolymer particles (TEPs) are planktonic, organic biofilms, these microgel particles are partly composed of poly- microgels that are ubiquitous in aqueous environments. Increasing saccharides with highly surface-active polymers of fucose and evidence indicates that TEPs play an active role in the process of rhamnose (16). They are, thus, about two to four orders of aquatic biofilm formation. Frequently, TEPs are intensely colonized magnitude stickier than phytoplankton or mineral particles and by bacteria and other microorganisms, thus serving as hot spots of have a high probability of coagulation or surface attachment upon intense microbial activity. We introduce the term “protobiofilm” to collision (17, 18). Therefore, TEPs are likely to play an important refer to TEPs with extensive microbial outgrowth and colonization. role in coating submersed surfaces and in the formation of aquatic Such particles display most of the characteristics of developing bio- biofilm (14, 15, 19). film, with the exception of being attached to a surface. In this study, Biofilm is defined as a sessile assemblage of complex microbial coastal seawater was passed through custom-designed flow cells microcolonies, attached to a surface, and held together within a that enabled direct observation of TEPs and protobiofilm in the matrix of self-produced, predominantly mucopolysaccharide EPSs feedwater stream by bright-field and epifluorescence microscopy. (20–22). The microcolonies are characterized by a basic architec- Additionally, we could follow biofilm development on immersed ture of multilayered, loosely packed bacterial cells encased in surfaces inside the flow cells. Within minutes, we observed TEP and EPSs, separated by interstitial water channels that allow transport protobiofilm patches adhering to these surfaces. By 30 min, confo- of nutrients, oxygen, chemical messengers, genetic material, and cal laser-scanning microscopy (CLSM) revealed numerous patches antimicrobial agents (22). Once established, biofilms are noto- of Con A and SYTO 9 staining structures covering the surfaces. riously resistant to removal by treatments with chlorination, Atomic force microscopy showed details of a thin, highly sticky, biocides, or antibiotics because of the protection provided by the organic conditioning layer between these patches. Bright-field multilayered EPS matrix. Therefore, much current, applied re- and epifluorescence microscopy and CLSM showed that biofilm de- search is aimed at inhibiting either the outgrowth of biofilm fl velopment (observed until 24 h) was profoundly inhibited in ow forming bacteria or bacterial adhesion to sensitive surfaces. fi cells with seawater pre ltered to remove most large TEPs and pro- These applied approaches are based on the following con- fi fi tobio lm. We propose a revised paradigm for aquatic bio lm de- ception of aquatic biofilm formation: an initial, preconditioning velopment that emphasizes the critical role of microgel particles phase, lasting from a few seconds to several hours, changes the fi such as TEPs and protobio lm in facilitating this process. Recogni- chemical and physical characteristics of the surface (23–25). fi tion of the role of planktonic microgels in aquatic bio lm formation Dissolved organic polymers and colloids present in the overlying can have applied importance for the water industry. water immediately begin to adhere to the surface, forming a thin (<300-nm) “conditioning film” composed of large variety of ransparent exopolymer particles (TEPs) are intensely sticky, adsorbed molecules: polysaccharides, proteins, lipids, and humic Torganic microgels, ranging in size from ∼0.4 to >200 μm, and nucleic acids (25, 26). Bacterial cells in the overlying water present in large numbers in all aquatic environments. Since first encounter the conditioning film and adhere to the surface. Cell described by Alldredge et al. (1), the ubiquity and multiple eco- adhesion is initially reversible, involving weak electrostatic forces system functions of TEPs have been extensively documented in and hydrophobic interactions. In this phase, bacteria still exhibit the oceanographic and limnological literature (2, 3). Brownian motion and are easily removed by application of mild TEPs and other microgel particles in marine and freshwaters shear forces. After several hours, most of the adhering bacteria are a part of a size continuum of organic matter that ranges from become irreversibly attached through strong dipole–dipole polymers through nanogels to microgels to very large marine (or forces, hydrogen and covalent ionic bonding, and hydrophobic lake) snow particles. Nano- and microsized porous gels composed interactions. The attached bacteria proliferate using dissolved of polysaccharides, proteins, and nucleic acids form from organic organic matter as a nutritional source and are triggered to pro- polymers and colloids in seawater by abiotic processes driven by duce EPSs, eventually forming mature biofilm (20, 27, 28). Fac- electrostatic and hydrophobic bonding (4–6). TEPs can also derive tors involved in the development of mature biofilm include directly from gelatinous envelopes surrounding algal cells, from bacterial quorum sensing (29, 30), nutrient availability (31), and bacterial mucous, or from degradation processes of marine or lake cell death and lysis (32). Depending on environmental condi- snow and other detrital material (7). Senescent or nutrient- tions, within hours to days after the initial irreversible adhesion, stressed algae and cyanobacteria have also been shown to gener- ate TEPs directly (8). Planktonic organic microgels such as TEPs “ ” may provide the scaffolding for hot spots of intense microbial Author contributions: E.B.-Z., I.B.F., and T.B. designed research; E.B.-Z. performed activity (9, 10). These gel clusters frequently harbor extensive research; O.G. contributed new reagents/analytic tools; E.B.-Z., I.B.-F., and T.B. analyzed populations of bacteria (11, 12) (in this paper, the term “bacteria” data; and E.B.-Z., I.B.-F., and T.B. wrote the paper. refers to both bacteria and archaea) and larger microorganisms The authors declare no conflict of interest. such as protista and algae (13). This article is a PNAS Direct Submission. Recently, TEPs have been implicated as an important factor in 1To whom correspondence should be addressed. E-mail: [email protected]. fi MICROBIOLOGY the development of aquatic bio lm (2, 14, 15). Like the extracel- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lular polymeric substances (EPSs) that form the matrix of microbial 1073/pnas.1203708109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1203708109 PNAS | June 5, 2012 | vol. 109 | no. 23 | 9119–9124 Downloaded by guest on October 5, 2021 the organized structure of a mature biofilm develops. The pro- cess described above posits that the critical step for the estab- lishment of biofilm is the successful, irreversible adherence of single bacteria to the substrate and assumes that the nutrition fueling bacterial growth in aquatic biofilm derives from dissolved organic matter within the overlying water. In the present study, we followed the initial stages (minutes to hours) of biofilm development using an experimental flow cell system (Fig. 1). Our results confirm the hypothesis that TEPs, in particular large TEPs heavily colonized by bacteria and other microorganisms that we have termed “protobiofilm,” play a crit- ical role in the initial stages of biofilm formation and significantly accelerate the rate of biofilm establishment. Based on the results of the present study, we propose a modified model of aquatic biofilm formation that takes into account the involvement of microgel particles such as TEPs in this process. Results and Discussion TEP and Planktonic Protobiofilm. Many studies have been published Fig. 2. Bright-field and epifluorescence overlay images of in situ planktonic on the occurrence and ecosystem importance in aquatic envi- protobiofilm (A and C) and uncolonized TEPs (B and D) visualized in sea- ronments of planktonic “hot spots” (2, 9, 10, 13). These are water passing through a flow cell. Bacteria (green) were stained with SYTO 9 generally visualized as clusters of microorganisms held on and and TEPs (blue) with Alcian Blue. Picophytoplankton (red) was identified by fl within a gel-like matrix and profoundly influence biogeochemical chlorophyll a auto uorescence. transformations within the water mass (10). Here, we propose “ fi ” the term protobio lm to refer to planktonic microgel clusters and C, may act as planktonic hot spots of microbial metabolism. within which extensive microbial outgrowth and colonization Within the confines of these heavily colonized microgel particles, have occurred. Such particles display most of the characteristics diffusion of signaling molecules released by prokaryote cells should of early developing biofilm, with the exception of being attached be greatly restricted, thereby