Microcystis is one of the most common toxic cyanobacterial genus able to proliferate worldwide in eutrophic freshwater environments (Preece et al., 2017). This genus forms colonies embedded in a mucilaginous matrix, and may proliferate into large floating biomass leading to ecological disturbances and representing a health risk for both humans and animals due to their capacity to produce toxins (Lance et al., 2010; Metcalf et al., 2012; Meriluoto et al., 2017). As nutrients and temperature are the main factors influencing cyanobacterial growth, the frequency, severity and duration of these mucilaginous Microcystis blooms are increasing due to eutrophication and climate change (O’Neil et al., 2012; Rigosi et al., 2014; Preece et al., 2017; Paerl et al., 2018).
The formation and maintenance of Microcystis in colonial form is conditioned to the presence of Extracellular Polymeric Substances (EPS) (Yang et al., 2008; Zhu et al., 2014; Chen et al., 2019; Xiao et al., 2019). Microcystis EPS take the form of a mucus envelope sticking cells to each other known as mucilage. These EPS affect significantly the surface properties of the cells by electrostatic binding (Liu and Fang, 2002) or polymer bridging (Vogelaar et al., 2005) and consequently, have a strong impact on Microcystis aggregation and colony formation (Le et al., 2022).
Cyanobacterial EPS occur in different forms and, based on their degrees of adhesion cell (Zhou Yang et al., 2008; Qu et al., 2012), may be classified into free EPS (other names found in the literature are soluble macromolecules, slimes and colloids) and attached EPS (or named as sheaths, loosely bound polymers, transparent exopolymer particles (TEP), capsular polymers, condensed gels, and attached organic materials) (Nielsen and Jahn, 1999; Laspidou and Rittmann, 2002; Sheng et al., 2010). Free EPS are weakly bound with cells or dissolved into the environment, while attached EPS are closely bound with cells (Wingender et al. 1999; Delattre et al., 2016; Cruz et al., 2020). However, it should be mentioned that the definition of these two fractions (free and attached) is, in many studies, unclear and depends significantly on the chosen EPS extraction methods (Frølund et al., 1996; D’Abzac et al., 2010; Bourven et al., 2011). Comparisons of the available methods have been performed and different extraction strategies have been tested and developed,especially for Microcystis samples (Xu et al., 2013; Liu et al., 2014; Huang et al., 2021). As an efficient and easy approach, low-speed centrifugation or filtration recovering supernatant or the filtrate is widely accepted to extract free EPS (Sheng et al., 2010; Pannard et al., 2016; Huang et al., 2021). For attached EPS, extraction methods with various physical and chemical protocols like ultrasound-assisted extraction or treatment with ethylenediaminetetraacetic acid (EDTA), cation exchange resin (CER), sodium hydroxide (NaOH) or formaldehyde have been proposed (Liu and Fang, 2002; Sheng et al., 2005; D’Abzac et al., 2010). More specifically for Microcystis, Xu et al. (2013) showed that the best method to isolate attached EPS was to heat the fixed cells at 60°C for 30 minutes in water.
Produced by different processes including excretion, sorption and cell lysis, EPS released by microorganisms are metabolic-excess waste products (Fogg, 1983; Liu et al., 2018). Mague et al. (1980) determined that phytoplankton usually excrete 5–20% of the carbon they fix which contribute to the nutrients and carbon pool, but more may be released under stress conditions (Fogg, 1983). The composition and production of EPS in cyanobacteria are both closely related to the taxonomy (Plude et al., 1991; Forni et al., 1997) and physiology (Xiao ans Zheng, 2016) of the species/genus studied and are therefore strongly influenced by abiotic and biotic factors such as light (Li et al., 2013; Srivastava et al., 2019; Wei et al., 2021), temperature (Duan et al., 2018), nutrients (Wang et al., 2011; Wang et al., 2011; Qu et al., 2018;), salinity (Ozturk and Aslim, 2010; Zhang et al., 2011; Wang et al., 2013), as well as predation (Yang et al., 2008; Cai et al., 2021). Among the above-mentioned abiotic factors, salinity strongly affects the expansion of mucilaginous Microcystis blooms to estuaries and along the freshwater-to-marine continuum (Preece et al., 2017; Paerl et al., 2018; Bormans et al., 2019; Sampognaro et al., 2020). However, the production and composition of EPS associated with natural colonies of Microcystis under salt stress remain unknown.
Indeed, very few studies have focused on the characterization of the composition of both free and attached fractions of EPS in cyanobacteria and in particular in Microcystis. Moreover, these studies were conducted with Microcystis strains under laboratory conditions where Microcystis exist predominantly as single cells (Li et al., 2013; Yang et al., 2008;). Consequently, the ecological role of EPS fractions on natural Microcystis colonies under different environmental conditions remains unclear.
In the present study, the production and composition of EPS from a unicellular Microcystis aeruginosa strain (PCC 7806) were compared with those from mucilaginous natural colonies of a cyanobacterial bloom dominated by Microcystis. To get further insight into EPS composition and in particularly the presence of proteins, lipids and polysaccharides, free and attached fractions of EPS were prepared and analysed. In a second time, we explored whether EPS production from the unicellular and the mucilaginous colonial form of Microcystis was impacted when cells were exposed to a salinity stress. Finally, we investigated the EPS production of the natural colonies under a combined salinity and nutrient stress condition (representing a land-sea continuum). We hypothesized that (1) EPS production would be higher for the mucilaginous colonial form than the unicellular form and (2) that the natural colonies would be more resistant to salt stress.