**2. Characteristics of EPS produced from** *Synechocystis*

The abbreviation EPS has variously been used to indicate extracellular polysaccharide, exopolysaccharides, exopolymers and extracellular polymeric substances. The material has been shown to contain nucleic acids, proteins, humic substances and lipid depending on its origin and environmental source. In addition, EPS may contain material derived from cell lysis and adsorbed materials that adhere to the natural polymers secreted from the surrounding environment. This adds to the complexity of EPS from a structural perspective. Most of this EPS is associated with the formation of aggregates or biofilms. The extracellular polysaccharide material varies in consistency, thickness and response to dye staining [32]. In laboratory culture, EPS is not an essential component, but in nature offers adaptive functionality. The characteristics of this material have been widely examined and there appears to be quite a large diversity in chemical composition and functionality. For example, in many cyanobacteria, this extra polysaccharide material plays a role in protecting cells from environmental extremes and stress. In certain strains, the release of exopolysaccharides together with sucrose and trehalose has been associated with desiccation resistance [33] and stabilization of cells when dried by air. In natural strains, dense layers may make strains less popular as food for predators relative to strains devoid of such material [34]. Attachment of benthic cyanobacteria to sediments, plant cells and other surfaces has been associated with extracellular polysaccharides and their hydrophobic nature [35]. It has also been proposed that secreted exopolysaccharides may play a role in precipitation of particles such as clay in aquatic environments clarifying the sur‐ rounding water. With precipitation more light is available for photoautotrophic metabolism [36]. The exopolysaccharides have also been proposed to disperse the cells themselves, facilitating optimum nutrient uptake [37]. In strains of *Microcystis*, the extracellular polysac‐ charide plays a role in metal ion accumulation, providing essential minerals for cyanobacterial metabolism. Such metals are often the few essential nutrients needed by photoautotrophic strains [38]. Protection of oxygen sensitive nitrogenase (used to fix nitrogen) has also been proposed as a function for attached extra polysaccharide material which can function by limiting oxygen transport to cells [39].

In addition to these important roles, extracellular polysaccharide plays a key role in cell aggregation and in biofilm formation [32]. As a major structural component of biofilm, EPS plays a role in allowing microorganisms to exist in large cell densities of mixed populations. This allows extensive communication to occur and exchange of genetic material via horizontal gene transfer. Participating cyanobacteria can thus adapt and evolve through the acquisition of genetic material from cells present in the biofilm community. In cell suspensions, EPS is distributed between the cell surface [in the case of capsular or cell-bound EPS] and the aqueous phase containing slime or free EPS, or as a hydrated matrix in biofilm with a composition that depends on growth phase and solution chemistry [40]. This mixture mediates adhesion and binding through interfacial processes including covalent or ionic bonding, dipole interactions, steric interactions, and hydrophobic association.

Many factors associated with EPS and surface layers of the cyanobacterial biomass can affect metal biosorption. pH has a major effect, where binding is decreased as a function of low pH, while other factors include whether the biomass is free or immobilised, the growth, age and metabolic state of the biomass, surface area of the cells or biomass for binding, the presence of competing ions in the effluent, the equilibrium binding concentrations, the flow rates, the nature of the metal complex and the temperature of the binding reaction. Thus there are numerous parameters that need careful attention to ensure optimal biosorption.

#### **2.1. Composition of the EPS and extrapolysaccharide material**

The *Synechocystis* genus is of interest in this respect because of its natural freshwater or marine habitat. In addition *Synechocystis* produces EPS (Extrapolysaccharide Substances) which can be either cell attached or released exopolysaccharides (RPS) [31]. This EPS can biosorb a range of material including metals which can be produced as a by-product of other biotechnological processes at scale. Thus while there may be many candidates for biomass biosorption, the sorption efficiency, scale and economics may well be key factors in adoption of a particular

The abbreviation EPS has variously been used to indicate extracellular polysaccharide, exopolysaccharides, exopolymers and extracellular polymeric substances. The material has been shown to contain nucleic acids, proteins, humic substances and lipid depending on its origin and environmental source. In addition, EPS may contain material derived from cell lysis and adsorbed materials that adhere to the natural polymers secreted from the surrounding environment. This adds to the complexity of EPS from a structural perspective. Most of this EPS is associated with the formation of aggregates or biofilms. The extracellular polysaccharide material varies in consistency, thickness and response to dye staining [32]. In laboratory culture, EPS is not an essential component, but in nature offers adaptive functionality. The characteristics of this material have been widely examined and there appears to be quite a large diversity in chemical composition and functionality. For example, in many cyanobacteria, this extra polysaccharide material plays a role in protecting cells from environmental extremes and stress. In certain strains, the release of exopolysaccharides together with sucrose and trehalose has been associated with desiccation resistance [33] and stabilization of cells when dried by air. In natural strains, dense layers may make strains less popular as food for predators relative to strains devoid of such material [34]. Attachment of benthic cyanobacteria to sediments, plant cells and other surfaces has been associated with extracellular polysaccharides and their hydrophobic nature [35]. It has also been proposed that secreted exopolysaccharides may play a role in precipitation of particles such as clay in aquatic environments clarifying the sur‐ rounding water. With precipitation more light is available for photoautotrophic metabolism [36]. The exopolysaccharides have also been proposed to disperse the cells themselves, facilitating optimum nutrient uptake [37]. In strains of *Microcystis*, the extracellular polysac‐ charide plays a role in metal ion accumulation, providing essential minerals for cyanobacterial metabolism. Such metals are often the few essential nutrients needed by photoautotrophic strains [38]. Protection of oxygen sensitive nitrogenase (used to fix nitrogen) has also been proposed as a function for attached extra polysaccharide material which can function by

In addition to these important roles, extracellular polysaccharide plays a key role in cell aggregation and in biofilm formation [32]. As a major structural component of biofilm, EPS plays a role in allowing microorganisms to exist in large cell densities of mixed populations. This allows extensive communication to occur and exchange of genetic material via horizontal gene transfer. Participating cyanobacteria can thus adapt and evolve through the acquisition

technology or species for an *in situ* industrial process.

54 Advances in Bioremediation of Wastewater and Polluted Soil

limiting oxygen transport to cells [39].

**2. Characteristics of EPS produced from** *Synechocystis*

Bacterial EPS can exist in many forms; as cell-bound capsular polysaccharides, unbound "slime", and as an O-antigen component of lipopolysaccharide [41]. EPS is generally observed as a sheath or capsule. This is a thin layer surrounding the cell membrane with concentric or radial fibres which vary in volume and layer composition. The material may also be observed as a slime layer, which is more loosely associated or as a soluble form which is released [42]. Much of the EPS or slime layers have limited association with the surface of the bacterial cell whereas capsular polysaccharides can be strongly connected to cell surfaces by means of a covalent attachment to phospholipid or lipid A molecules at the surface [43]. This division in nomenclature may become masked as capsular material is released and becomes free as a result of growth or leakage of the material into the growth medium.

In general, bacterial polysaccharides are composed of repeating monosaccharide units, forming homo- or hetero- polysaccharides linked via glycosidic linkages. Capsular polysac‐ charides are usually linear with molecular weights up to 1000 kDa. These are linked to a lipid anchor which in the classical *E. coli* system can be lipid A in the case of lipopolysaccharides. The chemical structure of lipid A is highly conserved, with the general backbone consisting of a β-1-6-linked disaccharide of 2-amino-2-deoxy-D-glucose to which fatty acids, often 3 hydroxyalkanoic acids, are linked by ester or amide linkages [41]. The inner region of the core oligosaccharide consists generally of 3-deoxy-D-manno-oct-2-ulosonic acid and L-glycero-Dmanno-heptose. These together with phosphate, and less frequently hexuronic acid and other sugar acid residues, give rise to the anionic nature of both the inner core region and of lipid A. The phosphate groups present often act to link amino alcohols to 4-amino-4-deoxy-Larabinose or other amino sugar residues present [41]. The complexity can be increased by the presence of stereoisomers, enantiomers, structural modifications of monosaccharide units and branching [44]. Table 2 illustrates the composition of EPS produced by bacteria. The exact nature of cyanobacterial EPS is largely uncharacterised but given the conservation of EPS composition in bacterial species, it can be assumed to be generally similar amongst cyanobac‐ teria. EPS in cyanobacteria is characterized by having relatively few sugar types consisting of the pentoses- xylose, arabinose and ribose; the hexoses -galactose, glucose (found in 90% of polymers) and mannose (found in 80% of polymers); and derived hexoses rhamnose (found in 80% of polymers), fucose, glucuronic and galacturonic acids, with occasionally methyl and amino sugars [32]. The presence of glucuronic and galacturonic acids associated with EPS further accounts for the anionic nature of many such polymers.

Amongst the cyanobacteria, there is quite a wide variation in the quantity of such polymeric material produced, varying from 144 mg. L-1 day-1 in the case of *C. capsulate* ATCC 43193 to 2 mg. L-1 day-1 in the case of *Synechocystis* PCC 6803 [45], which is amongst the lowest producer of this exopolysaccharide material. There have been few studies on whether compositional change occurs in such material. In *Synechocystis*, it has been shown that strains can modify the composition of this material, particularly as a function of culture age and in response to nutrients. In many cyanobacteria, the exopolysaccharide material may be a subcomponent of the extrapolysaccharide material that remains attached to the cyanobacterial cell itself and leaks or is physically broken away due to the habitat location, flow conditions or activity of the organism. This may occur as the extrapolysaccharide layers enlarge and may become solubilized or fractured as a natural part of the growth cycle. In the case of *Synechocystis*, the small amounts released suggest that it occurs at a low level and is not a common behaviour. In *Synechocystis*, neither addition of up to 0.5M of sodium chloride, nor glyoxylate, nor altering the light intensity during growth affected release of exopolysaccharides [45] suggesting that leakage occurred rather than an active release process. The components in *Synechocystis* PCC 6803 and PCC 6714 appeared different from other cyanobacteria. Substituent group analysis showed an absence of acetate or pyruvate with some sulfate substituent detected and a rather high protein content [46]. It had been thought that sulfated exopolysaccharide material was only found in eukaryotic algae so its presence in *Synechocystis* is somewhat unusual. Such sulfated material is thought to have antiviral activity and may be a defence strategy in natural environments [32]. Acetate groups have been proposed to hinder cation binding so their absence in *Synechocystis* is of significance in its potential role in metal binding. Extrapolysac‐ charide material with high levels of charged groups (such as sulfated derivatives) are impor‐ tant in forming polymeric matrices [47] with metallic ions and indeed natural clay particles being a prerequisite for good metal binding.


**Table 2.** Types of microbial surface compounds associated with biosorption.
