Production and characterization of extracellular carbohydrate polymer from Cyanothece sp. CCY 0110

Abstract

Cyanobacterial extracellular polymeric substances (EPS) are heteropolysaccharides that possess characteristics suitable for industrial applications, notably a high number of different monomers, strong anionic nature and high hydrophobicity. However, systematic studies that unveil the conditions influencing EPS synthesis and/or its characteristics are mandatory. In this work, Cyanothece sp. CCY 0110 was used as model organism. Our results revealed that this strain is among the most efficient EPS producers, and that the amount of RPS (released polysaccharides) is mainly related to the number of cells, rather than to the amount produced by each cell. Light was the key parameter, with high light intensity enhancing significantly RPS production (reaching 1.8 g L⁻¹), especially in the presence of combined nitrogen. The data showed that RPS are composed by nine different monosaccharides (including two uronic acids), the presence of sulfate groups and peptides, and that the polymer is remarkably thermostable and amorphous in nature.

Introduction

Cyanobacteria, like a wide range of other microorganisms, are able to synthesize and secrete extracellular polymeric substances (EPS) mainly constituted by heteropolysaccharides. These EPS can remain associated with the cell surface as sheaths, capsules and/or slimes, or be released into the surrounding environment as released polysaccharides (RPS). It has been hypothesized that they might be involved in the protection of the cells, the formation of biofilms, and/or the sequestration/immobilization of metal ions (Parker et al., 1996, Pereira et al., 2009, Sutherland, 1999). Cyanobacterial EPS have distinctive characteristics compared to those produced by other microorganisms: (i) contain generally 6–10 different monomers increasing the number of possible structural conformations of the polymer, (ii) have a strong anionic nature due to the presence of two different uronic acids and sulfate groups, and (iii) have high hydrophobicity conferred by the presence of ester-linked acetyl groups, peptidic moieties and deoxysugars (De Philippis et al., 2001, Shepherd et al., 1995). Altogether, these characteristics make cyanobacterial EPS very attractive for biotechnological applications such as the removal of heavy metal cations from polluted waters. In an industrial context, the use of cyanobacteria (or microbes in general) is advantageous compared to the use of plants or algae, as their growth rates are higher, the costs of production are lower (owing to their minimal nutrition requirements), and their growth conditions are easier to manipulate (Parikh and Madamwar, 2006, Pereira et al., 2009, Selbmann et al., 2002). Despite all these advantages, the characteristics of the polymers produced are strain-dependent and the number of systematic studies on the conditions that influence EPS production is still scarce, limiting the successful implementation of a biotechnological system based on cyanobacterial EPS (Pereira et al., 2009). Therefore, it is imperative to identify the most advantage EPS-producing cyanobacteria, as well as the culture conditions that influence the synthesis and/or the characteristics of the EPS produced. Previous studies indicate that the presence/absence of combined nitrogen, sulfur, glycerol and aeration, as well as different salt (NaCl) concentrations, temperatures, light intensities and light regimens are important factors to be considered, although no systematic studies were performed for a given strain (De Philippis and Vincenzini, 1998, Pereira et al., 2009). Once the conditions affecting EPS production are identified, the knowledge generated can be used to optimize the productivity and the desirable characteristics of the polymer.

Among EPS-producing cyanobacteria, several members of the Cyanothece genus are described as strong EPS producers (De Philippis et al., 1998, Micheletti et al., 2008a, Parikh and Madamwar, 2006, Shah et al., 1999). In addition, strains belonging to this genus have been isolated from both freshwaters and marine environments, allowing a given strain to be used for a specific purpose/environment. Nowadays, the study of marine strains is of extreme importance since it allows the exploitation of an abundant resource, saltwater, compared to the scarcity of freshwater.

In this work, a multidisciplinary approach was used to elucidate the conditions that influence EPS production by the unicellular marine N₂-fixing Cyanothece sp. CCY 0110, and to characterize the released polymer. Moreover, a scale-up was performed in a 10 L bioreactor to evaluate the potential use of this strain on an industrial scale.