The importance of ETS-05 in the context of therapeutic muds of the Euganean District led to the interest in analyzing the growth optima of this strain. ETS-05 represents in fact a source of high value compounds and could be exploited to both enrich the presence of the strain in the mud microbiota and to optimize its cultivation with biotechnological purposes. Considering the interest in EPS released by ETS-05, temperature, light spectra, light intensity, and nitrogen availability were evaluated not only to observe the culture response, but also to boost their production. Since EPS are obtained from the supernatant, intracellular high value compound production could be linked to the EPS one. Phycocyanin for instance was considered for its use in several fields (pharmaceutical, food, cosmetic (Saini et al. 2018)).
Effect of light spectra on growth was considered, knowing that red and yellow lights (de Mooij et al. 2016; Toyoshima et al. 2020) are generally more effective. Moreover Baer et al. (Baer et al. 2016) showed the importance to determine the optimized RGB mixture for every species. ETS-05 growth was not drastically influenced by light spectra, other than blue light that determined a drastic reduction in the biomass compared to other lights tested, as described in Synechococcus elongatus with 50 µmol photons m− 2 s− 1 (Ooms et al. 2017) and for Arthrospira platensis comparing red and blue light (Lima et al. 2018). According to Han and collegues (Han et al. 2015), both red and blue monochromatic lights enhanced EPS production in Nostoc flagelliforme, compared to white light exposure. However in our study, ETS-05 did not show improvements in EPS release using other lights rather than white light. Similar results were obtained for Nostoc calcicola RDU-3 with higher growth and polysaccharides release in white light followed by yellow, red, green, and blue lights (Singh and Das 2011). Some cyanobacteria can perform photoacclimation processes called chromatic acclimations (CA), involving he regulation of photosystems I and II and of phycobilisomes (Sanfilippo et al. 2019). Some Phormidium strains have been found, through bioinformatic analysis, to perform CA7, a modulation in the quantity of PEC according to the light spectrum (Hirose et al. 2019). Chromatic acclimation in red and green light was described in Phormidium sp. C86, with more synthesis of PE in green light and of PC in red light (Westermann and Wehrmeyer 1995). Alteration in PE and PC content was also observed in Phormidium autumnale CCAP1462/10 (Palinska et al. 2011). No change in pigmentation was observed in ETS-05, other than a diminished amount of all pigments in blue light, therefore the strain seems not able to perform any known kind of chromatic acclimation.
Thermotolerance of the species, anticipated considering its presence during the mud maturation process up to 47°C (Gris et al. 2020), determined higher biomass to be reached at 45°C. Higher temperature tested of 50°C led nonetheless to death of the organisms at 4 days, after a slight increase in OD750nm in the first days of cultivation. At the optimal temperature for biomass, higher EPS production was also obtained, as tested in cyanobacteria Scytonema tolypothrichoides and Tolypothrix bouteillei (Kvíderová et al. 2019). There was a direct correlation between temperature and EPS release. Same correlation was observed for PC up to 40°C where larger quantities were obtained: 221.8 ± 9.9 mg gDW−1. The content of PC in this condition was up to 21% w wDW−1, similarly to A. platensis (Xie et al. 2015), the most productive source of PC (Nwoba et al. 2019).
ETS-05 optimal light intensity corresponded to 100 µmol photons m− 2 s− 1 reaching a dry weight of 1.13 g L− 1 in 9 days. This was a promising result considering the 1.7 g L− 1 obtained for A. platensis cultivated for 15 days in raceways ponds (Raeisossadati et al. 2019). It is also possible that supplementing CO2 growth of ETS-05 could further increase. At lowest (25 µmol photons m− 2 s− 1) and highest (400 µmol photons m− 2 s− 1) intensities, biomass was reduced to a third or half. Direct correlation between EPS release and light intensity determined higher EPS concentration at 400 µmol photons m− 2 s− 1, indicating in this case the absence of a relationship between DW and EPS optima, as for T. bouteillei (Kvíderová et al. 2019). The positive effect of high light on EPS release was also observed in Cyanobacterium aponinum (Gris et al. 2017), Nostoc sp. (Ge et al. 2014a) and Microcoleus vaginatus (Ge et al. 2014b). Synthesis of phycobiliproteins behaved in the opposite way, with higher concentrations obtained at 25 µmol photons m− 2 s− 1, decreasing up to 10 mg gDW−1 at 400 µmol photons m− 2 s− 1. Chlorophyll a and total carotenoids content remained more or less similar in each intensity tested. There seems to be a trade-off between EPS and PC production, with optima conditions for one being non advantageous for the other.
Interestingly, under low and high light, ETS-05 filaments had different lengths, while other morphological features considered (cells dimension, shape, membrane thickness, internal arrangement) did not changed. We hypothesized that high light exposure stimulates hormogonia formation in ETS-05. Their differentiation can be stimulated or inhibited by environmental factors such as light and nutrients, in a species-specific manner (de Marsac 1994). As mentioned before, hormogonia are involved in the release of exopolysaccharides called HPS (Risser and Meeks 2013), to enhance the gliding motility of these trichomes for dispersal. Part of the quantified EPS in higher light intensities could be therefore correlated with abundance of hormogonia.
Finally, nitrogen depletion was tested since it is known to enhance EPS release in Nostoc sp. BTA97, Anabaena sp. BTA990 (Tiwari et al. 2015) Spirulina sp. (Nicolaus et al. 1999) and Cyanothece sp. 113 (Su et al. 2007). However, it was not observed a positive relation between the absence of this nutrient and EPS production in ETS-05. This condition was indeed too detrimental for growth and synthesis of compound of interest, even in a two-step cultivation system. Ultimately, it is important to notice the species-specific variability that characterize EPS production in cyanobacteria, implying the requirement to verify the best condition for each strain.
Furthermore, from an industrial point of view, using the residual biomass of EPS and hydrophilic pigment extraction as starting material, carotenoids, and lipids (not evaluated in this study) could be obtained. In (Liu et al. 2016) was for example successfully verified an integrated production of triacylglycerols and astaxanthin (as high-value carotenoid) using the microalgae Chlorella zofingiensis. Carotenoids from cyanobacteria can have various applications: anti-inflammatory, antioxidant, antitumor, color enhancer, anti-aging agent for cosmetics (Pagels et al. 2021). Furthermore, chlorophylls present biotechnological applications as food colorant, for cosmetics production and for human health being anti-inflammatory, antioxidant and antitumor agents (da Silva Ferreira and Sant’Anna 2017).
Bioinformatic analysis of putative genes involved in EPS assembly and secretion was performed to understand which mechanisms are carried out in ETS-05. Results highlighted the presence in ETS-05 genome of Wzy-dependent, ABC transporter-dependent (Pereira et al. 2015) and HPS-linked pathways (Zuniga et al. 2020a). Moreover other candidates were highlighted: proteins characterized by poly_export and SLBB domains, that can therefore act as OPX proteins. Real-Time qPCR allowed to verify the involvement of some of these putative genes: wzc (belonging to Wzy-dependent pathway), ps_ex (possibly coding for a OPX protein) and hrmK (probably involved in hormogonia formation), all augmented the expression when ETS-05 was exposed to optimal light, compared to low-light condition and the starting point.
To sum up, wzc, and ps_ex could be promising target genes to overexpress for an additional EPS assembly and release. On the other hand, enhancement of hormogonia formation through hrmK could stimulate HPS release but advantageous effects on biomass achievement should be further investigated.
Ultimately, the potential toxicity of ETS-05 was investigated, resulting in the absence of the genes involved in the synthesis of microcystin, cylindrospermopsin, saxitoxin, and lyngbyatoxin. These findings confirmed the absence of toxicity that was observed with an in vivo co-cultivation of ETS-05 and zebrafish (Danio rerio) larvae (Zampieri et al. 2020).
In this research ETS-05 was studied being the target species of the maturation process for the making of the Euganean therapeutic muds, possessing for this reason an importance in the territory and in the obtainment of this unique product with verified bioactive properties. Moreover, these results showed that ETS-05 could represent an interesting organism for biotechnological applications, considering the limited resources needed for its cultivation and the numerous high value compounds produced. In particular EPS synthesized by this species, which possess a demonstrated anti-inflammatory activity, can reach high yield when modulating the growth conditions. In addition to that, the absence of toxicity of the species is an important factor to consider in the optic of use its high value compounds for animal or human consumption and therapy. Finally, we investigated the involvement of several genes linked to EPS assembly and release by the organism with the hope to shed some light on this process that should gain more interest in the near future. The correlation between these genes’ expression and the condition of growth tested is a promising starting point for further characterizations and possible manipulations of ETS-05 to boost EPS productivity.