The term ‘biofilm’ was coined long ago (Costerton et al., 1987), and refers to microbial cells that have attached to a surface or aggregated with each other, and have secreted a gelatinous matrix of EPS. The ability of a microbial cell, such as a bacterium, to attach, secrete EPS and form a biofilm under laboratory conditions, is well-established. The secretion of EPS (by cells) is a key emergent property of the biofilm (Flemming and Wingender, 2010; Flemming et al., 2016); the property that directly influences adaptations that cells utilize to enhance their efficiency and survival. The secretion of an EPS matrix represents, in the broadest sense, an extension of the cell. The presence of EPS facilitates the self-organization of cells into localized communities, and provides biofilm cells with an enhanced capability for: trapping other organics and localizing their digestion by extracellular enzymes, coordinating cell–cell chemical communication [quorum sensing (QS)], facilitating gene-exchange, and provides a degree of physical stability. The EPS often form a localization matrix for other molecules, keeping them in spatial proximity to cells where they can be efficiently utilized.

It is now generally recognized within microbiology that the ‘biofilm state’ is an omnipresent feature of microbial flora in most environments (Hall-Stoodley et al., 2004). Biofilms occur under a wide range of conditions and environments, and whose influences span aquatic, terrestrial, the epi- and endo-biont communities of plants and animals, which can be commensal, symbiotic or pathogenic. The cells within a biofilm can move, and periodically reorient themselves in relation to one another, and in doing so can resist invasion by other cells (Houry et al., 2012). The EPS matrix of biofilms provides a three-dimensional architecture framework that allows the arrangements of cells movements relative to other microbes as well as positioning among sharp geochemical gradients (Decho, 2000b). This will not be discussed further here, but directly contributes to the remarkable plasticity of biofilm cells. The EPS form a matrix of largely anionic molecules near cells, affording them with a proximal environment that is more stabilizing, and conducive to manipulation by the cell (Table 1), and one that contributes to broader ocean processes. However, in this overview we will not discuss biofilms as systems, except with regard to their secretion of EPS.

Finally, it is important to note that in ocean systems, the microbial communities of aggregates suspended in the water-column, and the sea-surface slick communities of oceans are also biofilms, since these communities contain EPS, and exhibit differing levels of organization. EPS are also secreted as a ‘metabolic by product.’ These are most apparent during the later stages of phytoplankton blooms, and will be discussed further below. Taken together, microbial extracellular secretions are now thought to comprise a large portion of the bioavailable carbon pool in oceans, especially in dissolved forms. The total amount of microbially produced EPS, although difficult to measure accurately and precisely, is likely to be very substantial.

Dissolved organic carbon in the ocean can be classified into three broadly defined pools of carbon based on their turnover times: labile, semilabile, and refractory. Collectively, the concentrations of these three DOC pools typically range from 60 to 90 μmol/L in the upper ocean water column, decreasing with depth to 40 μmol/L in the deep sea (Hansell, 2002). Fluctuations in the concentration of DOC in the water column occurs over certain periods of the year, due largely to periods of elevated photosynthetic production since this is the main source that fuels each of these three classes of DOC. The combined effect of biological and physical processes that alter the concentrations of these three pools of DOC is represented in the size-reactivity-continuum model (Amon and Benner, 1996; Benner and Amon, 2015), for which microbial processing of these molecules is the major mechanism that leads to rendering them progressively more recalcitrant (Jiao et al., 2010).

From the standpoint of microbial cells, the EPS, especially when in a gel state, form a three-dimensional matrix or scaffold within which cells can orient themselves relative to one another. The presence of certain polymers can afford the matrix physical stability. Polymers such as amyloids and/or eDNA (discussed below) can serve as an architectural framework for the EPS. Each type of EPS component can offer different physical properties (Chew et al., 2014). The physical ultrastructure of the EPS matrix has been very difficult to image in a fully hydrated state (Decho, 1999), owing to its delicate nature. Excellent pioneering efforts have been conducted by Dohnalkova et al. (2011) using a unique cryo-TEM approach. Recent developments in cryo-TEM and -SEM may provide further insights into this complex matrix.

Bacterial and microalgal EPS exist in nature in a range of different physical states, most of which are operationally defined. Capsules consist of polymers that closely surround individual or multiple cells and often serve a protective role (Whitfield, 2006). Further away from cells, EPS can exist as tight, dense-gels, to a continuum of physical states from looser-slime to truly dissolved forms. Dissolved forms, in the absence of cells, may condense to microgels in the open ocean (Chin et al., 1998; Verdugo et al., 2004; Verdugo and Santschi, 2010); a process that is of significant importance and has been well-summarized in a review (Verdugo, 2012). While differences in these physical states are relatively arbitrary, they likely serve very different functions to the microbial cells secreting them. The physical state of EPS results from a combination of the polymer concentrations, types and abundances of ions, composition, and steric availability of functional groups on polymers. Recently, this has been reviewed in greater detail (Neu and Lawrence, 2016).

Some microbial EPS can exhibit significant viscoelastic properties, being able to stretch and retract in response to an applied force such as intermittent water flow (Fabbri and Stoodley, 2016). This property offers the microbial cells contained within a biofilm with a certain degree of physical flexibility and mechanical resiliency (Peterson et al., 2015). This flexibility is important to the ability of cells to persist on a surface or as an aggregate in the water-column.

Compositional information is a useful starting point for investigating EPS. Strictly speaking, the term EPS is an operational designation that refers to the milieu of ‘larger’ molecules contained in the extracellular matrix in proximity to cells (Wotton, 2004). Many of the molecules contained in natural EPS cannot be conveniently characterized as either protein, lipid or carbohydrate. For this reason, it has been called the ‘dark matter’ of biofilms (Flemming et al., 2007). It is now known that the EPS matrix can be quite heterogeneous, especially over small spatial scales (see Microdomains within EPS Matrices). It is for this reason that analyses of ‘bulk’ samples will not capture the smaller-scale variability that are critical to understanding the physical and chemical properties of the in situ matrix. When EPS from natural biofilms, for example, are extracted and then reconstituted, the physical (e.g., gel, viscocity, rheology, etc.) properties of the reconstituted EPS often do not readily resemble those of the original biofilm. (In much the same way, a cell cannot be taken apart via extractions, and then put back together as a functioning cell.) This suggests that the molecules within the matrix may have important molecular organization either by purposeful design or by result of the environment. It is important from a functional standpoint, to determine which molecules and molecular interactions contribute to physical properties such as gel formation, rheology, and diffusion-slowing, and chemical properties, such as sorption.

Finally, while matrix is actively secreted by cells, the properties of EPS may be changed post-secretion, modified by geochemical, enzymatic, and photochemical processes, and additionally contain trapped or sorbed molecules. These modifications may have dramatic effects on their physical properties. Thus, EPS under natural conditions exist in a ‘continuum’ of compositional and partial degradation states. Thus, it is imperative that future non-destructive approaches (e.g., Raman spectroscopy) are developed for characterizations of EPS in situ.

Laboratory studies of bacteria secreting EPS show that cells typically produce sugar monomers that are exported, then assembled to the existing polymer outside of the outer membrane (Whitfield, 2006; Sutherland, 2016). Polymers may consist of single sugar monomers, called homopolymers, or consist of several monomers linked together to form a repeating unit, called heteropolymers. Since several types of repeating units may be generated, this provides the cell with the capability to alter the physical chemical properties by mixing different amounts of repeating units. By changing the building blocks (i.e., repeating units), the polymer composition can be varied. This allows the polymer to have a variable composition and provides the cell the capability to modify its extracellular polymers in response to changing conditions.

At present, there are no specific biosignatures that can be assigned reliably for detection of EPS, simply because similar glycol-based compounds are produced throughout biological systems for many purposes. The secretion of carbohydrates and other glycosylated polymers is not unique to bacteria or even microorganisms; rather, it is a universal biological strategy employed by microalgae, fungi, invertebrate and vertebrate animals, and plants (Underwood and Paterson, 2003).

Extracellular polymeric substance have often been considered synonymous with ‘exopolysaccharides’ and acronym EPS. This was partially based on the carbohydrate-focus of investigators at the time, and additionally due to the artifact of carbon-rich culture conditions that were used to grow bacteria and obtain abundant quantities of EPS. Much has been learned regarding polysaccharide chemistry from these seminal studies. While polysaccharides are a major component of many natural EPS, they are only a component. The EPS matrix is now known to contain several different major groups of molecules, whose roles and involvement are still under study, and will be discussed briefly below. Hence, much discussion has evolved about what exactly constitutes the EPS matrix. Three points emerge as one studies the EPS of natural systems: (1) Many different types of molecules interact to provide the physical structure, impart chemical properties, and even actively manipulate EPS properties for the cell; (2) EPS-compositional studies should not be limited to culture-based systems; and (3) EPS under natural conditions will likely exist in a continuum of partial-degradation states.

Diffusion is a key process in the design of the microbial cell, as it is the primary means by which small organic molecules and ions may be taken up by cells. Diffusion is also of relevance in the movement of signal molecules (i.e., autoinducers) involved in QS, extracellular enzymes, and antimicrobial agents. EPS can be broadly considered a sorptive sponge for the binding, trapping and concentration of organics and ions (Decho, 1990). Over the small spatial scales of biofilms, the EPS matrix influences the diffusion process. Diffusion is a multi-faceted process that is influenced by temperature, ionic concentrations, etc. A major driver in diffusion, of course, is the relative concentration gradient (Brogioli and Vailati, 2001). The density and properties of the EPS can influence diffusion rates (of ions or molecules) so they can range negligible (compared to diffusion in pure water at the same temperature) to having significantly slowed diffusivities (Decho, 2015).

Although bulk measurements of diffusivity may be estimated, it is difficult to determine how the smaller-scale variability in EPS densities influence diffusion at these scales. The natural matrix of EPS within aggregates or surface biofilms is often filled with channels, which by microbial design or by environmental influence, enhances mass transfer to/from cells.

A number of investigators have carefully measured diffusion rate constants by monitoring the movement of fluorescent molecules over time using confocal scanning laser microscopy and other approaches (Lawrence et al., 1994; Guiot et al., 2002; Stewart, 2002; De Beer et al., 2004; Waharte et al., 2010; Neu and Lawrence, 2014). Lawrence et al. (1994) initially used fluorescent molecules and confocal scanning laser microscopy to examine diffusivities and found them to be variable, ranging from those of pure water (d = 1.0) to extreme diffusion-slowing effects (d = 0.02) by the matrix. From a practical standpoint, one can assume there will be variability within an aggregate or attached biofilm. Collectively, these studies have shown that considerable changes occur in the movement of molecules and ions over microspatial distances (i.e., μms), which can relate to the observed heterogeneity that occurs within biofilms (Stewart and Franklin, 2008).

Physical trapping of organic and inorganic colloids, and nanoparticles also occurs in the EPS matrix. The viscoelastic nature and the dispersed arrangements of EPS at the surface-most fringes of biofilms make them ideal for the physical trapping of colloids, small particles, and/or sorption of ions and molecules. Sorption is influenced by a number of factors. These include pH, the forms and concentrations of ion(s), and the type(s) of ligands (binding sites) and associations (e.g., ionic- and covalent-bonds, van der Waals forces, etc.). The pH can have a strong effect on ionic binding, especially with regard to many EPS (Braissant et al., 2007, 2009; Gutierrez et al., 2008). In general, acidic pH tends to inhibit ion binding, while neutral or basic pH tend to promote binding. A caveat is that not all ions bind equally. A second caveat is that certain functional groups bind ions more efficiently at a given pH. For example, as the pH rises to near neutral, more complexation may occur to carboxyl sites. The increase in binding of divalent cations often results in a more cohesive polymeric gel structure.

This pH-dependent sorption process has practical importance in ocean systems (and biofilms) because the most abundant divalent ions in seawater are Ca²⁺ and Mg²⁺. These ions often form suitable (bi-dentate) bridges between adjacent EPS molecules having carboxylic acid groups, and can contribute to gel formation or floc (marine snow) formation in the water column. However, other important ions (at a given pH) may ‘outcompete’ Ca²⁺ and Mg²⁺ for binding sites. The binding of transition and other metals, such as Th, Cd, Cu, Ag, Fe, and Se, to EPS isolated from different environments, such as hydrothermal vents, microbial mats, and other areas, has been described (Schlekat et al., 1998; Zhang et al., 2008; Moppert et al., 2009; Deschatre et al., 2013). Metal binding to EPS of surface floc material (i.e., marine snow) in the surface waters of oceans, and subsequent sinking of flocs may result in significant vertical transport (flux) of trace elements to ocean floor, a process of global biogeochemical significance (as mentioned above).

While ocean seawater is often pH 7.8–8.2, the range of pH over smaller spatial (and temporal) scales can be quite dramatic. In microbial mat systems, where highly active bacterial respiration and photosynthesis occur, the pH has been shown to vary from pH 6.0–10.0 over a 24 h (i.e., diel) cycle (Visscher et al., 2000; Des Marais, 2003; Baumgartner et al., 2006). This is due to net photosynthesis during daylight (which raise pH), and net respiration during darkness (which lowers pH). This can result in regular diel changes in the complexation of ions, and hence influence the physical stability of EPS over a 24 h cycle. The diffusion-slowing properties of EPS contribute to the sharp geochemical gradients often observed within aggregate and attached biofilms (Baumgartner et al., 2006).

The biofilm is an organic gel coating (of EPS) on a surface (or within a suspended aggregate) with a collage of cells, and sorbed or localized molecules and ions, colloids, and particulates. All of these different components, individually or interactively, will influence the optical properties (i.e., refraction, scattering, and absorption of photons) of the broader surface (or water). The EPS can be thought of as a “semi-translucent” gel having different densities. Several processes act in concert to alter the optical properties of sediments. First, the polymers themselves appear to decrease the reflectance of the surface. In sediment systems, this can alter the amount of light entering the sediments. This is due to a combination of two processes. First, the gel polymers increase the spacing between sediment grains. This allows more light to enter in the spaces between grains, rather than being reflected from closely packed sediments. Second, the gel state of the polymer acts as a ‘photon trap’ because it mediates a change in refractive index, relative to seawater. This enhances the forward-scattering of photons, relative to back-scattering. This has been termed the ‘biofilm gel-effect’ (Decho et al., 2003). The functional value is that light may be more homogenously scattered around photosynthetic cells, and allow cells to conduct photosynthesis deeper in sediments (or mats).

As light interacts with a surface, photons are either reflected, scattered, refracted and/or absorbed. Reflectance involves back-scattering of photons at a fixed angle (relative to incident direction of photon). Often photons are ‘scattered’ at many angles relative to the incident. Refraction involves continuing through the surface, but altering the angle (relative to the incident) resulting in a change in refractive index. Absorbance involves the capture of photon energy by surface molecules or atoms. Absorbed photons may be re-emitted, as fluorescence (within pico-sec to nano-secs after absorbance), or released as heat. However, the biofilm is not simply a translucent gel but rather a three-dimensional matrix harboring cells, sorbed, or localized molecules (e.g., scytonemins, amino acids, etc.), colloids, and particulates. Biological chromophores (molecules that absorb light near specific wavelengths) include the purines and pyrimidines of DNA, the ‘ringed’ amino acids (tyrosine, phenylalanine, etc.), and other molecules. The sea surface layer is known to harbor EPS gels (Wurl and Holmes, 2008). Of special interest will be how the sea-surface layers of EPS influence photon penetration into the underlying water.

Do microbial communities communicate and coordinate activities? Classical microbiology during much of the past century has taught us to understand microbes simply as individual cells. Recently, however, a growing body of evidence supports the idea that bacteria often act in groups, rather than as individuals. When bacteria are attached, their proximity to each other results in the development of interactive relationships ranging from antagonistic to agonistic, and even altruistic. These interactions are often chemically mediated but are tempered by the ever-changing conditions of their local environment. The diffusion-slowing properties of the EPS matrix facilitates the development of such relationships among cells in a way that cannot be accomplished by free-living planktonic cells.

Quorum sensing is a type of bacterial cell–cell communication that involves the exchange of chemical signals among nearby cells to coordinate behaviors that are best conducted in groups (Fuqua et al., 1996). It involves the production, detection, and response by cells to diffusible signaling molecules (i.e., autoinducers). Autoinducers accumulate in the proximal environment as the bacterial population increases. When autoinducer concentration reaches a threshold-level, cells collectively alter gene expression. Many group activities such as bioluminescence, antibiotic production, and EPS secretion (Camilli and Bassler, 2006) are regulated by QS.

Quorum sensing can also be utilized by cells for ‘diffusion-sensing’ (Redfield, 2002). This allows bacteria to sense the diffusional properties of its proximal environment, presumably to ‘make decisions’ whether to conduct more metabolically costly processes, such as production and release of extracellular enzymes, plasmids, antibiotics, etc. (Ruparell et al., 2016). Together, these two processes, quorum- and diffusion-sensing, have been termed ‘efficiency sensing’ (Hense et al., 2007).

The foundation for cell–cell cooperative interactions originally was proposed for explaining the bioluminescence by a marine luminescent bacterium, previously Photobacterium, renamed Vibrio fischeri, and then Aliivibrio fischeri, which was isolated from a small Hawaiian squid (Ruby and Nealson, 1977; Nyholm et al., 2000). Studies progressively showed that autoinducer molecules, upon reaching a threshold concentration in the medium, triggered changes in gene expression that resulted in bacterial luminescence. Luminescence by populations of symbiotic bacteria, localized in the light organs of the squid, afforded it a selective advantage against predation. The ability to communicate, coordinate, and act as groups, however, does not relinquish the cells as an individual unit. Microbial cells can (and do) still act as individual cells. This amazing flexibility likely contributes to the tremendous success and resiliency of bacteria.

In open surface-water ocean environments, QS can have large-scale effects, especially when in overwhelming abundances. An obvious example of this was the ‘milky ocean’ that was observed at night by satellite off of Somalia, Africa (Nealson and Hastings, 2004; Miller et al., 2005). The milky ocean, which was 100s of square km in size, was due to QS-triggered bioluminescence in ocean surface populations of bacteria.

Several different classes of chemical signals exist. Most were described from the study of infection-causing bacteria. These include acylhomoserine lactones (AHL), unique oligopeptides, furanosyl borate diesters (Autoinducer-2), and gamma-butyrolactones (Waters and Bassler, 2005). The AHLs comprise a class of approximately 18 different types of signal molecules that are released into the surrounding environment, and eventually bind to an intracellular receptor protein, whose complex then triggers changes in gene expression (Churchill and Chen, 2011). AHLs in marine environments have been found in sponges (Taylor et al., 2004), microbial mats (McLean et al., 1997; Decho et al., 2009, 2010) and marine snow (Hmelo et al., 2011). Interestingly, signals such as AHLs are prone to inactivation under certain environmental conditions such as high pH (>8.0) (Decho et al., 2009; Hmelo and van Mooy, 2009). It is not known how fluctuating conditions (e.g., pH, oxidants, desiccation, photocatalytic degradation) in natural environments influence chemical signaling and coordination of microbial activities (Horswill et al., 2007; Decho et al., 2009, 2011; Frey et al., 2010). Signaling, however, will likely be localized and most pronounced within EPS matrices, and within planktonic aggregates (Gram et al., 2002; Wagner-Dobler et al., 2005; Hmelo et al., 2011; Amin et al., 2015; Jatt et al., 2015).

Signaling using AHLs, for example, is not limited to heterotrophic bacteria. Rather it has been found in photosynthetic cyanobacteria (Sharif et al., 2008) and Archaea (Zhang et al., 2012). Importantly, it is now realized that this form of cell–cell communication can occur in single-species population, but may also be utilized by inter-Kingdom consortia, such as plant-microbe and animal–microbe associations. Contrastingly, molecules that may act as signals for some bacteria, may act as antibiotics against other bacteria (Kaufmann et al., 2005; Davies et al., 2006; Schertzer et al., 2009; Johnson et al., 2016). A final note is that currently only several classes of signaling molecules (e.g., AHLs, AI-2, and peptides, diffusible signal factors (DSFs), etc.) are known (Schaefer et al., 2008; Papenfort and Bassler, 2016, for review). However, QS and similar interactions via chemical signaling are likely to occur using a variety of signals; most of which may be unknown at present.

Bacteria possess the capability to bud-off portions of their cell membranes (Schooling et al., 2009; Biller et al., 2014), which are then released as extracellular vesicles. The vesicles provide bacteria with the ability to package molecules within a surrounding lipid membrane, and release them in their surrounding extracellular environment, and localize them within the EPS matrix. This can provide a protective ‘minefield’ against antibiotics, preserve extracellular signals and plasmids, and provide other functions as well. The presence of extracellular vesicles within biofilms, and specifically the EPS matrix, is now realized to be quite common (Mashburn and Whiteley, 2005; Biller et al., 2014). The vesicle composition is often similar to the plasma membrane (Gram-positives) or outer cell membrane (in Gram-negatives), but additionally contain specific proteins as part of the vesicle. The vesicles can package a wide range of molecules such as eDNA, RNA, e-enzymes, antibiotics, and signal molecules, and likely provide protective effects for the packaged molecules they carry (Mashburn-Warren et al., 2008; Schooling et al., 2009).

Gene exchange is an important process among bacteria. The EPS matrix can enhance gene exchange among cells for several reasons. First, conjugation (i.e., a uni-directional exchange of plasmids via a pilus connecting two cells) requires extended contact for a prolonged period of time (approximately 20 min). In open-water systems, this is difficult due to Brownian motion constraints. When localized in a three-dimensional EPS matrix, two cells can remain relatively stationary for prolonged periods of time, which can facilitate conjugative gene exchange. Second, extracellular DNA, used in transformation, can be rapidly degraded once outside of the cell, or strongly sorbed to sediment particles. When DNA is immobilized within EPS, its persistence can be enhanced, and thus increase chances for transformational exchange of DNA among cells. Direct measurements of these two processes, to our knowledge, are not yet available.

Degradation of organic matter and its mineralization to CO₂ is a fundamental process of bacteria. In many ocean environments, bacteria produce e-enzymes to partially hydrolyze organic matter that becomes sorbed or trapped by the EPS (Hoppe et al., 2001). When conducted efficiently, with minimal loss to the surrounding water, the biofilm can be an efficient external digestion system for the microbial community (Flemming and Wingender, 2010).

The localization of e-enzymes is a process that is important in open water aggregate- as well as attached-biofilms. In order for efficient diffusional uptake, both enzymes and their hydrolysis products must remain localized in proximity to cells (e.g., approximately 30 μm). EPS provide a matrix to localize both enzymes and their hydrolysis products relatively close to cells. It is not known, however, how e-enzymes remain localized within the EPS matrix. Are they attached (bonded) to polymers with active sites exposed? In studies of other systems, bacteria are known to localize polysaccharases and other e-enzymes (Sutherland, 1999, 2016). e-Enzymes are also known to be contained within extracellular vesicles, localized within the expolymer matrix (Mashburn and Whiteley, 2005; Mashburn-Warren et al., 2008; Elhanawy et al., 2014), and e-DNA nucleases were found in Vibrio cholerae biofilms (Seper et al., 2011). Indeed, elevated microbial activities, such as enzymatic activities, have been reported in marine snow particles at higher-levels than those in surrounding sea water (Smith et al., 1992; Ploug et al., 1999; Grossart et al., 2003; Jatt et al., 2015), thus suggesting that marine snow are hotspots for remineralization of organic and inorganic materials (Azam and Long, 2001; Thornton et al., 2010).

Insight has been provided through studies of other systems (Tielen et al., 2013). They showed that extracellular lipase was protected against heat denaturation via complexation with the EPS alginate. Using molecular modeling they were able to show that e-enzymes can be physically bound to EPS, however, the enzyme/EPS bond must occur away from active sites on the enzyme. Finally, this bonding provides enhanced stability against denaturation. However, questions remain, such as: how are enzymatic activities maintained outside the cell? Empirical evidence has been relatively limited. Do the functional equivalents of extracellular chaperones help to maintain activities (i.e., prevent denaturation) of e-enzymes? It is also not known how extracellular enzymes may modify the EPS themselves.

Mats are well-known for their association with the biogeochemical precipitation of minerals such as carbonates (e.g., calcite, aragonite) (Decho, 2010). Present-day examples of precipitating mats include tufa mats, marine stromatolites, and marine thrombolites (see Dupraz et al., 2009, for review; Glunk et al., 2009; Tourney and Ngwenya, 2014). There are several different mechanisms known to directly or indirectly influence precipitation within mat environments (Visscher and Stolz, 2005). Microbial communities drive the basic alkalinity engine, which when coupled to the organic matrix of mats, results in biogeomineral precipitation (Dupraz and Visscher, 2005). Activities of several microbial groups, such as cyanobacteria, sulfate reducers, and anoxygenic phototrophs, can ‘promote precipitation,’ while other groups (e.g., aerobic heterotrophs, sulfur oxidizers, and fermenters) can ‘promote dissolution’ (Dupraz et al., 2009). Precipitation of CaCO₃ occurs in seawater that is near or exceeding supersaturation of carbonate ions, and has basic pH conditions (Arp et al., 2001, 2003). Cyanobacterial activities, for example, will raise the pH during daylight photosynthesis, which favors localized carbonate precipitation (Gautret et al., 2004; Ludwig et al., 2005). Specific moieties on EPS, such as acidic groups, can act as nuclei for subsequent CaCO₃ precipitation (Braissant et al., 2003; Bhaskar and Bhosle, 2005; Obst et al., 2009). Even bacterial cells themselves can serve as nucleation sites for precipitation (Varenyam et al., 2010). EPS can bind substantial amounts of free Ca²⁺ (Braissant et al., 2007) and other minerals such as phosphate and sulfate (Gallagher et al., 2013) from the surrounding water. This can result in precipitation of EPS-associated minerals such as apatite [Ca₅(PO₄)₃(F,Cl,OH)], struvite (MgNH₄PO₄⋅6H₂O), dolomite [CaMg(CO₃)₂], and aragonite (Gallagher et al., 2012, 2013).

Under some conditions, however, EPS can inhibit precipitation, or even contribute to carbonate dissolution. Cation-binding by EPS removes free Ca²⁺ ions from solution, through depletion of carbonate minerals from the proximal surroundings. Acidic amino acids, such as aspartic or glutamic acids, and carboxylated polysaccharides (i.e., CO32- groups of uronic acids) can act as strong inhibitors of CaCO₃ precipitation (Kawaguchi and Decho, 2002a,b; Dupraz et al., 2004; Gautret and Trichet, 2005). The functional groups and their steric availability (to bind ions) are key in this process (Rieger et al., 2007; Yang et al., 2008). The role(s) in carbonate precipitation of specific microbial clades such as sulfate reducers, however, remains controversial (Meister, 2013), but likely involves different types of community interactions under normal marine, hypersaline, and alkaline conditions (Gallagher et al., 2014).

Marine stromatolites are microbial mats having repeating layers of precipitated micritic laminae produced through interaction of the microbial communities and the environment (Krumbein, 1983). In present day, they occur in only a few limited marine environments such as the Bahamas (Reid et al., 2000; Paerl et al., 2001) and Shark Bay in Western Australia (Goh et al., 2009). Studies extending for over a decade have examined present-day open-water, subtidal, marine stromatolites at Highborne Cay (Bahamas) and showed the surface microbial community consisted of several distinct mat stages (i.e., termed Types 1, 2, and 3), each having very different phenotypic characteristics (Reid et al., 2000). The EPS produced during these stages had very different properties and influenced the microbial communities within. In the Type 1 stage, the community exhibited “high growth.” It consisted of dense cyanobacteria with high EPS production that grew (upward) quickly, consuming resources (Decho et al., 2005). The abundant EPS resulted in a “sticky” surface that trapped ooid grains (i.e., sediment) washing over the mats during high wave actions; a process that propagated the continued upward growth of the mat. The EPS contained ligands to chelate much of the available free Ca²⁺ ions. The net result was that EPS in the Type 1 mat inhibited CaCO₃ precipitation (Visscher et al., 2000). When a Type 1 mat transitions to a Type 2 mat, sulfate-reducing bacteria (SRB) increase in their relative abundances. EPS, initially produced by the cyanobacteria, are consumed by SRBs, then re-secreted as different EPS, a process which enhances localized precipitation (Visscher et al., 2000; Decho et al., 2005). Infared (FT-IR) spectral analyses of EPS extracted from the precipitate closely resembles those extracted from SRB mat isolates (Braissant et al., 2009). Notably, the EPS properties change in the Type 2 mat, becoming “less sticky.” These studies illustrated how the EPS properties of the different mat could influence their properties and growth. The Type 2 mat resembles a classic microbial mat, with less EPS production (or accumulation), and little/no upward growth. The cyanobacteria, SRB, sulfur-oxidizing bacteria (SOB), and aerobic heterotrophs become spatially organized, and exhibit a closer metabolically coupling.

With rising levels of atmospheric CO₂, efforts are beginning to examine if certain carbonate-precipitating bacteria can be used to sequester (and store) CO₂ (Paul et al., 2017) and understand how microbial mat processes influence C storage (Bouton et al., 2016).

Sediment fluxes in marine systems are affected by many parameters, including sediment grain sizes, physically cohesive muds, and biologically cohesive microbial extracellular polymers (Gerbersdorf et al., 2009; Grabowski et al., 2011). EPS concentrations in marine sediments vary considerably (Underwood et al., 1995, 2004). EPS, especially those from microphytobenthos (e.g., diatomaceous mats), are important in cohesive sediment stability, and resistance against erosion and resuspension (Grant and Gust, 1987; Paterson, 1989; Smith and Underwood, 1998, 2000; Tolhurst et al., 2002; Underwood and Paterson, 2003; Hanlon et al., 2006). Levels of EPS present in sediments can be a crucial variable to sediment stability (Malarkey et al., 2015). However, more EPS isn’t always better. Interesting experimental studies by Paterson et al. (2008) showed that lower levels of EPS were more efficient in increasing erosional thresholds than abundant EPS conditions. While is well-established that the cohesive properties of EPS contribute to sediment stability, it is however not well-understood how the molecular-scale interactions of EPS themselves contribute to their cohesiveness (Paterson et al., 2008). Sediment-inhabiting small animals may indirectly influence sediment stability. For example, increased EPS production was shown to occur in the presence of a grazing nematode (Hubas et al., 2010). Initial studies suggest that QS (see above) may be involved in biofilm formation in certain diatoms (Yang et al., 2016) and offers the possibility that QS may contribute to the sediment stabilization process.

Finally, the very same bedform patterns that contribute to sediment stability in present-day sediments (e.g., ripples) also are considered as fossil evidences of sediment stabilization. These include patterns such as microbially induced sedimentary structures (MISSs), which are considered to be indirect fossil remnants that illustrate the very earliest vestiges of microbial mat life through geologic time (Noffke et al., 2001, 2013; Noffke, 2010).

It was realized early on that biofilms, and more-specifically their EPS, can represent a potentially labile carbon source for animals ingesting microbial cells (Decho, 1990). Since many small animals, present in both the water column and sediments of ocean systems, ingest microbial flora as a food source, they will coincidently ingest the closely associated EPS during the feeding process. Many invertebrate taxa are filter-feeders (i.e., straining suspended particles from the water) or deposit-feeders (i.e., ingesting sediments and their organics), therefore will consume microbial flora and their associated EPS as a food. Initial feeding experiments addressing EPS utilization by marine animals were conducted using EPS that were isolated from bacterial cultures grown in the presence of radioactively labeled (¹⁴C) substrates. Once the EPS were separated from cells and residual label, EPS were mixed with sediments and fed to animals. Results indicated that EPS comprised a highly labile carbon food source. Examples of such studies involved copepods (Decho and Moriarty, 1990), polychaete worms (Decho and Lopez, 1993), bivalves (Harvey and Luoma, 1985), and sea stars (Hoskins et al., 2003). Biofilms are even known to be grazed upon by benthic foraminifera (Bernhard and Bowser, 1992).

Extracellular polymeric substance can act as a ‘sorptive sponge.’ This is due to their ability to bind metals, other ions, and even relatively hydrophobic organic contaminants such as pesticides, which is attributed to the presence of charged moieties (i.e., positive or negatively charged functional groups) or hydrophobic moieties. This can result in the efficient trophic-transfer of metals and pesticides to consumer animals that are ingesting EPS. Together, these moieties serve to sorb and/or trap, and concentrate environmental contaminants. When animals ingest the matrix, coincidentally during their feeding on sediments, cells or flocs, they will consume the sorbed metals and organics as well. EPS have been shown to be an efficient trophic-transfer vehicle for sorbed metals in amphipods (Schlekat et al., 1998, 1999, 2000; Selck et al., 1999), and organic compounds such as pesticides, although most of the latter work has even been conducted in freshwater systems (Widenfalk et al., 2008; Lundqvist et al., 2010, 2012).

Virtually any type of surface, when placed in seawater becomes fouled with organisms ranging from bacteria to animals and algae; a process known as biofouling (Lewin, 1984). Bacteria and other microbes are generally the initial colonizing organisms of a surface. The presence of a bacterial biofilm often sets the stage for subsequent larval settlement (Tran and Hadfield, 2011). Understanding how biofilms interact with larval settlement is important to the broader biofouling process, which constitutes a costly ocean-engineering problem.

Studies in marine systems suggest that larval settlement is also a multistep process, which involves initial sensing of specific chemical cues, initial settlement and “tasting” of the surface, and finally more-permanent settlement. The initial sensing step of waterborne cues is a concentration-dependent process.

Presently, data suggest that in many cases, the larval settlement cues are molecules that are produced by adult conspecifics and are concentrated within the surface biofilm matrix or, in some cases, may be produced by the biofilms themselves (Unabia and Hadfield, 1999; Bao et al., 2007). Cue(s) can be multifunctional, acting as agonists, antagonists, or toxins (Ferrer and Zimmer, 2012; Guezennec et al., 2012). Behaviors and responses of larvae to cues are often species-dependent.

Settlement cues may be localized within the biofilm, and more-specifically by the EPS matrix. Diatom biofilms and the possible involvement of heat-stable settlement cues are involved in the settlement of the polychaete Hydroides elegans (Lam et al., 2003). Hydroides sp. are examples of the initial colonizers of open surfaces in warmer water regions. Once they are set, the complexity of colonizing species increases. Settlement cues may be produced by the biofilm itself but has been challenging to verify. The beneficial effect of a specific epibiotic bacterial biofilm on marine animal or plant hosts has been suggested (Holmström et al., 1992; Tran and Hadfield, 2011). Coralline red algae, for example, are highly inductive surfaces for the settlement of marine invertebrates, and are now realized to be strongly influenced by the surface microbial flora and their cues (Nielsen et al., 2015).

In contrast, the inhibition of larval settlement seems to be influenced by waterborne or biofilm-associated molecules. This is important to the potential control of biofouling. Early studies noted that many marine animals and macroalgae exhibit reduced biofouling, and suggested that chemical defenses may be involved (Holmström et al., 2002; Rao et al., 2007). More recently, studies have shown that specific chemical inhibitors can be produced by either the host organism (e.g., algae, animal) or by biofilm bacteria growing on the surface of the host (Lau and Qian, 2000). These have included both large and small molecules. In tunicates, proteins produced by biofilm bacteria inhibit further colonization by bacteria (Holmström et al., 1992). In a series of landmark studies, de Nys et al. (2009) showed the macroalga Delissia pulchra, was not subject to biofouling. This inhibition (of biofouling) operates by jamming the cell–cell chemical communication pathways of bacteria. As mentioned above, many gram-negative bacteria use AHLs in chemical communication. Release of specific AHL analogs, which are similar in molecular design can “jam” the QS pathway(s) of AHLs. Small halogenated furanones, resembling AHLs, interfered with chemical signaling in bacteria. An ecological function of antifouling molecules produced by plants and animals was postulated (and was tested) by Kjelleberg and colleagues (Franks et al., 2006). Harder et al. (2002) found large (>100 kDa) polysaccharide-containing molecules, produced by both a host macroalga (Ulva sp.) and Vibrio sp. bacteria, to inhibit larval settlement. These molecules act as a broad-spectrum inhibitor for settlement. This has touched off substantial exploration for chemically based inhibitors of biofouling in both nature and medicine by many laboratories. This infers the complex interaction in biofouling among host organisms, bacterial biofilms, and chemical cues. Since the biofouling of marine surfaces has both positive and negative effects to hatcheries, this area has an emerging impact on aquaculture processes (Joyce and Utting, 2015; Camacho-Chab et al., 2016). Finally, work is in progress to understand how climate change may affect processes such as larval settlement (Whalan and Webster, 2014).

In polar regions, metabolic processes are slowed by relatively cold temperatures. It is here that microbes also employ EPS to their advantage. Earlier, pioneering studies in Antarctic systems showed the presence of specific ‘anti-freeze proteins’ (i.e., glycoproteins) within the blood plasma of fish (Devries, 1971). The proteins would bind to ice crystals as they formed and prevent further growth of damaging ice crystals in the blood. This realization launched many subsequent studies of other organisms, including bacteria.

In Arctic and Antarctic systems, the presence of EPS play key roles as cryoprotectants, for attachment to sea-ice interfaces, and to survive enclosure in ice (Underwood et al., 2010, 2013). Studies of bacterial isolates from sea-ice systems have demonstrated that certain glycoproteins and exopolysaccharides act as cryoprotectants, which inhibit ice crystal nucleation, in addition to securing the attachment of cells to the ice surface (Nichols C. A. et al., 2005; Nichols C. M. et al., 2005; Marx et al., 2009; Ewert and Deming, 2014). EPS and TEP become routinely embedded in sea-ice (Meiners et al., 2003; Collins et al., 2008) and contribute to the survival of microbial cells in these environments (Krembs et al., 2002, 2011; Liu et al., 2013; Boetius et al., 2015). EPS, including TEP can account for the majority of the carbon pool in sea-ice, which is later released during melting (Miller et al., 2011; Wurl et al., 2011), and can even make their way into atmospheric ice (Wilson et al., 2015).

Since their initial discovery in 1977, ocean hydrothermal vent systems have received much scientific attention. They are located near specific regions of the ocean spreading centers of tectonic plates, where geothermally heated fluids, enriched in minerals, hydrogen sulfide, ammonia and methane are released and mix with much colder surrounding seawater. Mineral deposits form as chimneys, and are surrounded by islands of intense biological activity, where chemosynthetic bacteria and archaea form the base of a food web having a diversity of often unique animals (Tunnicliffe, 1991). For these reasons, they have been considered as a possible site for the origin of life on Earth, and as an analog for study in the exploration for possible life elsewhere.

Isolates of bacteria from vent systems demonstrate the capacity for abundant EPS production, perhaps to sequester dissolved minerals and other metals from the surrounding water (Raguénès et al., 1997a,b; Guezennec et al., 1998; Rougeaux et al., 1999, 2001; Guezennec, 2002). EPS, derived from isolate cultures, typically have uronic acid contents as high as 40%, and relatively high molecular masses (Guezennec, 2002). It is not yet understood, however, how the EPS may influence the microenvironment of the bacteria and archaea, in terms of e-enzymes, 3D-microspatial development of their communities, and microspatial acidification (to solubilize metal ions). It is not known if the EPS matrix facilitates these processes, and actually may serve to inhibit their precipitation, similarly to those in some shallow-water carbonate environments?

Hypersaline systems, such as salt ponds, salterns, and hypersaline lagoons, contain well-developed microbial mats. Many of these systems occur in proximity or directly connected to ocean systems, while others are inland. Examples of hypersaline systems are numerous and a few include Salt Pond, San Salvador, Bahamas (Pinckney and Paerl, 1997); Guerrero Negro, Baha California Sur, Mexico (Ley et al., 2006); Laguna Tebenquiche, Salar de Atama, Chile (Fernandez et al., 2016); Don Juan Pond [McMurdo Dry Valleys, Antarctica (Dickson et al., 2013)]; Dead Sea (Oren, 1994); Solar Lake, Sinai, Egypt (Teske et al., 1998); Hamelin Pool, Shark Bay, Western Australia (Goh et al., 2009); Polynesian islands (Rougeaux et al., 2001; Richert et al., 2005; Moppert et al., 2009).

The hypersaline environment presents unique challenges to microorganisms, especially in terms of fluctuations in ion concentrations and osmolarity. In addition, many hypersaline mats are exposed to intermittent and/or progressive desiccation. A lack of available water, during the desiccation process, can kill a bacterial cell, largely through denaturation of proteins and destabilization of cell membranes (see Potts, 1994, for review). EPS are an abundant component of such mats (Benninghoff et al., 2016), and likely provide a degree of protection to mat microbial flora against ion fluctuations and desiccation (Potts, 1994; Shaw et al., 2003; Decho, 2016). Interestingly, studies have shown that bacteria can survive in a desiccated state in salt crystal for 250 my (Vreeland et al., 2000).

In hypersaline mats, EPS occur in the form of capsules surrounding individual cells, or a larger EPS matrix surrounding many cells in a biofilm, which can buffer cells against either dessication or rapid changes in water potential. Salinities in hypersaline ponds are often >300 g/L (e.g., seawater is approximately 32 g/L), but can reach as high as 440 g/L, often with concentrations of individual ions not matching those observed for typical seawater. Here, mat communities often experience extended periods (i.e., days to months) of desiccation that often is followed by a rapid rehydration due to seasonal rain events.

Selective saltation also plays a role in the ability of mats to cope with increasing salinities. This allows less-soluble minerals to be removed from solution, as a function of concentration. The evaporation process that occurs throughout the dry season serves to increase ionic concentrations and promotes the selective precipitation of salts on the mat surface. As ionic concentrations increase, there is a sequential salting-out occurring of less-soluble minerals. For example, much of the Ca²⁺ is typically removed as gypsum (CaSO₄ 2H₂O) or smaller amounts of calcite (CaCO₃). Gypsum begins to precipitate when salinity concentrations reach about 160 ppt. At very high ionic concentrations (>300 ppt) NaCl begins to precipitate.

Some EPS may condense with increasing salinity, and even form a hydrophobic barrier on the surface of the biofilm. This may result in enhanced protection during subsequent desiccation. It has been proposed that the exclusion of ions occurs via the EPS matrix in response to increasing salinity, which is designed to reduce osmotic stress and conserve water within the mat (Decho, 2016). Desiccation is a process occurring in many areas of marine environments. On the fringes of ocean systems, specifically on the upper reaches of rocky intertidal zones, intermittent desiccation is a common process. The roles of EPS in stabilizing microbial communities require further investigation.