Cyanobacterial Exopolysaccharide (EPS) Synthesis and Metal Sequestration by Biosorption

Pinaki Hazra [State Aided College Teacher]
Dr. Gargi Saha Kesh [Associate Professor]

Abstract: Cyanobacteria produce extracellular polymeric substances (EPS) that are mainly made by high-molecular-mass heteropolysaccharides, with variable composition and their roles depend on the microorganism and the environmental conditions.  Cyanobacteria have the major roles to become an industrially important source of functional biopolymers. Their exopolysaccharides (EPS) consist of various types of chemical complexity, which predicts bioactive potential. Although some are reported to excrete large amounts of polysaccharides, others are still to be discovered. This review organizes available information on cyanobacterial EPS, including their composition, function and their heavy metal sequestration capacity . Compared to various types of conventional heavy metal removal methods, heavy metal removal by cyanobacteria  is a potential method, as it is a low cost method, in situ operable, and simple chemistry related. They are excellent machines for operation of multidirectional metal sequestration as they can sequester metal simultaneously through biosorption and bioaccumulation. Biosorption is a cell surface method, whereas bioaccumulation occurs within the cell. This study reviewed how cyanobacteria are able to absorb heavy metal ions by these two methods from an ambient water body and the protective machinery of cyanobacteria against heavy metal-induced toxicity. Further, among the different components of the cyanobacteria’s cell wall, this blue–green algae is able to  absorb the metal ion mainly through Exopolysaccharide (EPS).

Keywords: Industrial Activity ,Mining ,Agricultural Activity, Sewage Water & Natural Activities.

Introduction:

Heavy metals are foreign particles that are able to deteriorate the surface and groundwater quality and are toxic at low concentrations [1,2]. Heavy metals have a high density (∼5 g/cm3 ) and non-biodegradable [3], which transfer to receiving watershed by various processes such as industrial activity ,mining ,agricultural activity, sewage water  and, natural activities (weathering and erosion of bedrocks) [4]. When these metals enter the aquatic system, various biochemical reactions affect aquatic organisms and associated trophic levels, mainly when metals exceed the upper limit (1-2000ppm based on metal ions) and bioaccumulate [5]. Once marine species ingest those heavy metals up to the threshold limit, they show their effects on biological functions such as enzyme inhibition, degeneration of fatty acid, byssus formation. Heavy metals mainly chromium, nickel, vanadium, cobalt, and arsenic function as redox catalysts and generate free radicals. Many metal-induced diseases occur in fish, which include spiral deformity, blackening of the tail region, change in metabolic activity, and cellular intoxication resulting in death [5]. Finally, when humans consume these toxic metal-rich plants and fishes, different health diseases and carcinogenic effects are formed in humans by bioaccumulating these metals. Microbes' use to remove toxic metals has gained the spotlight over chemical methods because of their small size and a high surface area to volume ratio. Therefore it provides a large surface area for metal binding. Recent studies regarding the remediation of oil spills in the ocean have justified the advantages of bioremediation over chemical remediation techniques [12].

`Cyanobacteria have a very diverse taxonomic group and are found both in unicellular or multicellular forms that may be coccoid or filamentous [6]. These prokaryotic organisms have a bacterial-like cellular envelope. Their structure is similar to Gram-negative bacteria, but some features and thickness of peptidoglycan are the same as Gram-positive bacteria. In nature, cyanobacterial EPS can play various types of functions such as adhesive materials, structural, protection against environmental stress, bio weathering processes, gliding motility, and nutrient depositors in phototrophic biofilms or biological soil crusts [7].

Cyanobacteria are able to remove toxin from wastewater by a process called phycoremediation [8]. Organisms under cyanobacteria phyla play a unique role that make them an excellent tool for the biodetoxification of the heavy metal polluted water body. For example, the cell wall contains variety of multifunctional chemical groups (COO-, OH- etc.) and uniform metal absorbing sites inside cell (Metallothionein, Phytochelatins and, polyphosphate),  high removal efficiency, economically suitable and excellent retention power. Those properties help to metal ion sequestration through adsorption and accumulation procedures [9]. Although the presence of heavy metals in water may exert impact on the physiological processes of cyanobacteria, these cyanobacteria are also able to  adapt strategies at the cellular as well as the molecular level to bypass the stress generated by heavy the metals [28].

Chemodiversity ; 

The EPS in cyanobacteria is part of a complex network of extra polymeric substances, and it can also comprise proteins, nucleic acids, lipids, and secondary metabolites [10]. More than 13 various types of monosaccharides have been selected from C5 to C6 and underpinned on 40–50 glycosidic linkage tightly correlated to polysaccharide flexibility [9]. Glucose is found as a most common monosaccharide, however, some EPS strains were found to contain many other monosaccharides such as rhamnose, xylose, arabinose, fucose, mannose, and uronic acids [11]. Uronic acid have an exclusive presence in the cyanobacteria, being identified with a frequency of one or two units. Monosaccharides can be grouped by their form: hexoses (glucose, galactose, mannose, and fructose); pentoses (ribose, xylose, and arabinose); deoxyhexoses (fucose, rhamnose, and methyl rhamnose); acidic hexoses (glucuronic and galacturonic acid). The EPS of Arthrospira platensis strain exhibited rich EPS diversity, the CPS fraction the most diverse (fucose, galactose, glucose, mannose, rhamnose, ribose, and xylose) [12]. Methyl, pyruvyl, and succinyl groups can be present as well as sulfate groups, which are only found in archaea and eukaryotes. Additional types of monosaccharides such as N-acetyl glucosamine, 2,3-O-methyl rhamnose, 3-O-methyl rhamnose, 4-O-methyl rhamnose, and 3-O-methyl glucose are also reported [13]. At the macromolecular scale, these polymers are characterized by high molecular weight, which can range from kDa to MDa, whereas more than 75% of those characterized are heteropolymers [6]. The physico-chemical role of these building blocks is extremely rich. Although some exhibit hydrophobic character, which increases adhesion to solid surfaces, others are hydrophilic, sticking to minerals, nutrients, and water molecules. The combination of both moieties can promote an amphiphilic character allowing cyanobacteria to react in different ways to the surrounding environment [14]. Cyanoflan, an RPS isolated from the marine Cyanothece sp. has a high intrinsic viscosity and emulsifying activity in aqueous solutions [15]. Cyanobacteria constitute a prolific source of EPSs with physico-chemical properties. As a consequence of these natural complexities, a pitfall for structural elucidation burdens the number of available structures [9].

There are various possible fields of application for these polymers: (1) in the food, cosmetic, textile or painting industries, for the modification of the flow properties of water, i.e. as thickening, suspending or emulsifying agents[34] (2) in the pharmaceutical industry, because of their antiviral or immuno-stimulating properties or the capability of slowly releasing drugs [34] (3) in wastewater treatment plants or the goldsmith industry, for the chelation of toxic or valuable metal ions from water solutions, i.e. as biosorbents

Defense mechanism of cyanobacteria against metal-induced toxicity

When heavy metal ions get inside the cell, they induce lots of physiological stress due to the excess of the cell’s metal storage capacity. Heavy metal ion-induced toxic effects on cyanobacteria have different forms, as they can block functional groups of important molecules, generating cellular reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, singlet oxygen, hydroxyl radical [9], induce cell damage and successively lead to death .To fight metal induced toxicity, cyanobacteria have different strategies. As we mentioned earlier, the first line of defense is EPS. The negative functional group in EPS composition sequester a large amount of heavy metal ions [10]. When heavy metal ions get inside cyanobacteria by membrane transporter proteins, cyanobacteria begin to synthesize metal binding peptides (Metallothionein and phytochelatin) to detoxify metal ions [17]. Another mood of defense is expressing antiporter membrane proteins inside the cell, which transport back free metal ions outside the cell [18]. Besides all of these defense mechanisms, if free metal ions remain in cyanobacterial cells, these will induce metal ion toxicity by producing different biological or biochemical complexes [25].

Mechanism of heavy metal sequestration by cyanobacteria:

 Microorganisms are able to remove or biotransform different pollutants [19]. Microorganisms uptake toxic metals by using absorption (extracellular) and adsorption (intracellular) mechanisms. The extracellular absorption process is the active, metabolically driven process,  named bioaccumulation, and the passive is not a metabolically driven process (intracellular), known as biosorption.

Biosorption :

At physiological pH, toxic metals are absorbed by cyanobacteria via metabolic independent passive uptake called biosorption. The cell wall of bacteria is negatively charged, resulting in ionic interactions with positively charged heavy metals from the surrounding environment [20]. Negatively charged chemical groups are present both in live and dead organism’s cell surfaces. Hence, by the biosorption process, dead and live cellmass can consume heavy metal ions simultaneously [21]. Although metal sequestration by dead biomass is rapid [21],once metal ions are translocated into the cell, Cyanobacteria are able to transform heavy metal ions into harmless form during translocation. Those biotransformations of heavy metal ions by cyanobacteria are mediated by extracellular precipitation, valence conversion, or volatilization [18]. Biosorption is also done by cell surface peptidoglycan receptors functional groups by different mechanisms [22].

Mechanism 1: Peptidoglycan COO^- group is a good source for providing excellent adsorption surfaces for heavy metals. Adsorption is done by binding metals to membrane proteins, lipids, lipopolysaccharides, and exopolysaccharides [23]. Among these, exopolysaccharides (EPS) play a major role in sequestering metals by the action of uronic acid as it has high anionicity [24].

Mechanism 2: Translocation metal ion within the cell membrane happens when there is a metal ion concentration difference between the outer and intracellular environment [25]. Active transport of metal ions in the cell carried out by transporter proteins. These ions compete for multivalent carrier binding sites of transporters or, low molecular weight thiol binding happens [26].

Mechanism 3: Binding to chelating proteins is another active transport [24]. Cyanobacteria produce metallothionein (Chelating protein) in the cytoplasm, which are thiol rich cysteine residues complex. They aid metal ion sequestration when metal ions get inside the cytoplasm [24]. Besides metallothionein, many other metal-binding compounds such as phytochelatins and polyphosphates bind metal ions and detoxify them [26]. They form organometallic complexes, that are transferred to cell compartments such as vacuole, for storage. This process maintains cytoplasmic ion concentration and detoxification [27]. Among these mechanisms, EPS mediated metal ion sequestration is found to be higher. EPS structure and composition favour the sequestration of metal ions [27]. This polymer has been comprehensively studied as a treatment regime for reducing heavy metal contamination because of this property. To understand the mechanisms behind the metal ion sequestration through EPS, it is essential to know how the bacterial cells biosynthesize these polymeric substances.

Mechanism of Cyanobacterial exopolysaccharide (EPS) synthesis and factors effect on metal sequestration by biosorption

Biosorption of metal ions on the cyanobacterial outer cell is aided by EPS, which is a high molecular weight naturally occurring polymer. Characterization shows that EPS contains different polysaccharides, proteins, lipids, nucleotides, and secondary metabolites having a wide range of negative functional groups (Carboxylate, sulfate, sulfhydryl, amide, amine, and uronic acid) which provide binding sites for metal ions [28]. Most of the EPS are synthesized by cyanobacteria and exported to the extracellular environment. There are four types of enzymes responsible for the production of EPS.

a. Hexokinase is responsible for the phosphorylation of glucose (Glc) to glucose-6- phosphate (Glc-6-P) [29];

b. Uridine-5`-diphosphate (UDP)-glucose pyrophosphorylase mediates the conversion of Glc-1-P into UDP-Glc [30];

c. Glycosyltransferases (GTFs) are responsible for transporting sugar nucleotides to a repeating unit attached to glycosyl carrier lipid [31] ;

d. The last group of enzymes (Polymerase) is responsible for polymerizing the macromolecules outside the cell membrane [32]. The production of EPS in different pathway systems is the exploitation of cyanobacteria producing polysaccharides with good antiviral activity has not been considered worth developing new drugs. This is due to the long and very expensive procedures needed for the commercialization of new pharmaceutical products. The possible use of exopolysaccharide-producing cyanobacteria for the recovery of valuable metals from industrial wash waters seems to be more promising than most of the above-mentioned applications. Indeed, the high economical value of the metal, which can be easily recovered from the biosorbent, might justify the investment necessary for the production of the biomass. However, this field of application is still in its infancy and needs more research to establish a simple and cheap technology for the production and utilization of the cyanobacterial biomass as biosorbent, as well as for the recovery of the metal.

Several factors responsible for Cyanobacteria mediated heavy metal sequestering, such as rate of  growth , biomass, carrying capacity , contact time , pH  and temperature [33] .Light intensity and glucose concentration are able to influence the composition of the cellular wall, and thus biosorption capacity. The cells of the same microalgal species, cultured under different conditions of light intensity and glucose concentration, are found to have different metal biosorption characteristics [24]. The functional groups participating in the metal-binding process by the EPS-producing cyanobacteria may create a difference from strain to strain [21]. Chojnacka et al. found that different chemical groups may participate in metal ions binding at different pH. For example, at pH 2–5 carboxyl group, at pH 5–9 carboxyl and phosphate group, at pH 9–12 carboxyl, phosphate, and hydroxyl (or amine) group seem to be involved in metal sequestration from water. Synechococcus sp are able to remove different metal ion at pH 5-7.5 [25]. The contact time for reaching equilibrium for removal of metal ions from water are found to differ for different strains. The saturation of metal removal capability was reached within 5–6 h with C. capsulata and Nostoc, whereas with Spirulina platensis [21] or Synechococcus sp, the equilibrium was reached within 60–120 min. Conversely, for some strains like Tolypothrixceytonica [32] and Chlorella vulgaris [31], it is reported to require 14–15 days to remove metal from water efficiently. All cyanobacteria strains are found to sequester metal ions at around 25–30°C. Prior to make the cyanobacteria mediated metal removal process feasible for industrial application, provision must be made to regenerate the biomass for reuse. Lowering the pH (1-2) of the metal-loaded biomass suspension causes desorption of heavy metal cations by protons .

Although cyanobacteria mediated heavy metal removal is a cost effective method, there are some limitations. It is an important fact that cyanobacteria-mediated metal removal processes are slower than traditional chemical processes [33]. Further, cyanobacteria can also create a threat to the aquatic ecosystem, as some cyanobacterial species are toxic [20]. Hence, before applying cyanobacteria, one needs to identify toxic species such as Anabaena circinalis, Cylindrospermopsis raciborskii, Microcystis aeruginosa and Planktothrix sp. To overcome this situation, dead toxic Cyanobacteria may be used for heavy metal removal [22].

 Conclusions 

we have discussed  how EPS may enhance the metal binding capacity of cyanobacteria. However, further study is required on EPS producing cyanobacteria for more EPS production with genetic engineering. Those improvements may promote  cyanobacteria for more viability in a wide range of environmentally  adverse atmospheres.

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