**1. Introduction**

Cyanobacteria are a group of prokaryotic microorganisms found in fresh and marine waters and soils [1] and represent the first colonizers in drylands [2]. They have been studied for contributing to agricultural productivity in a process called "cyanobacterization", which consists of cyanobacteria inoculation in the soil to provide soil structural stability and biofertilizer properties [3–5]. The selection of feasible species for a given environmental condition, allied to a cheaper biomass production and dispersion strategy, represents a new biotechnological approach to increase fertility in agricultural soils.

This biotechnological potential is due to the formation of biocrusts, which are composed of microorganisms aggregated to soil particles [6,7]. Due to the excretion of exopolysaccharides (EPS), the biocrust-forming cyanobacteria allow: (i) the increase in soil organic carbon pool, and stimulation of exoenzyme activity in the soil [8]; (ii) water retention and infiltration, which have a positive correlation with the total carbohydrate content of the biocrust [9,10]; (iii) the retention of metabolites and nutrients, thus preventing

**Citation:** Moia, I.C.; Pereira, S.B.; Domizio, P.; De Philippis, R.; Adessi, A. *Phormidium ambiguum* and *Leptolyngbya ohadii* Exopolysaccharides under Low Water Availability. *Polymers* **2023**, *15*, 1889. https://doi.org/10.3390/ polym15081889

Academic Editor: Paschalis Alexandridis

Received: 27 January 2023 Revised: 31 March 2023 Accepted: 12 April 2023 Published: 14 April 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

molecules from leaching down to the subcrustal layer [11]; and (iv) the release of vitamins, amino acids and phytohormones in the soil, which act as biofertilizers [12,13].

The rather complex monosaccharidic composition usually provides to the cyanobacterial EPS an amphiphilic character, determining their functions in soil [14–16]. For example, the hydrophobic character of some fractions of the polymer, mainly due to the presence of deoxysugars, such as rhamnose and fucose, may favor the adhesion of the cyanobacterial filaments to solid substrates and the formation of soil aggregates [5,14,17]. On the other hand, the negatively charged, hydrophilic fractions of the EPS, characterized by a large presence of uronic acids and sulfate groups, are involved in binding minerals, nutrients and water molecules, thus favoring the survival of the microbial community residing in the biocrust [18,19]. These EPS features provide sustainment to the biocrusts and can enable cyanobacteria to cope with periods of desiccation [8,20,21].

The EPS versatility and their protection role confer resistance also for biofilms to reach ecological success [22]. For instance, the EPS hydrophobic/hydrophilic character was reported for biofilm-forming cyanobacteria isolated from wastewater treatment plant and brackish lagoon [23]. These authors reported that the high uronic acids proportions could be successfully exploited for different biotechnological applications. Moreover, Keshari et al. (2021) [24] showed that the cyanobacterium *Scytonema mille*, which has a thick sheath (polysaccharide anchored to the cell wall) was the major microorganism in the biofilms on the Buddha statue in India, indicating that the sheath may support the organism to cope with desiccation [24].

The abiotic conditions of the environment and the cyanobacterium species also influence the molecular size distribution of the EPS [9]. For example, the presence of exopolysaccharides with low molecular weight (MW) was more frequently present when the filamentous cyanobacterium *Leptolyngbya ohadii*, grown as biocrust, was subjected to high water availability [17]. This pattern might be related to the increase in the activity of enzymes that decompose the higher MW to smaller MW polymers [17]. Additionally, it was reported that *Phormidium ambiguum* liquid culture (where it experiences optimal nutritional conditions) and biocrusts produced polymers with MW distributed heterogeneously through the EPS fractions [25,26]. Therefore, the chemical and macromolecular characteristics of exopolysaccharides can be modulated according to soil type [25], nutrient supply [27], and water availability [17,21].

Recently, it has been shown that *L. ohadii*, isolated from biocrusts of the Negev desert [6,28], can revive after hydration/dehydration cycles [29,30]. The genus *Phormidium* has been found in arid areas, where hot deserts are present [31–33]. These areas are susceptible to wind erosion, due to the changes in physicochemical properties and decrease in the stability of the soil, that accelerates land degradation [34,35]. Therefore, it is important to evaluate cyanobacteria tolerating long periods of drought in order to stabilize and increase the strength of the soil. For instance, *P. ambiguum* was shown to perform an essential role in the aggregation of soil particles, improving soil structure and stability, and indicates it to be a good candidate to be used in soil restoration in arid areas [25,36]. However, little is known about these cyanobacteria growth and EPS composition and role upon low water availability when cultivated in different modes of life (sandy soil biocrusts and biofilms liquid cultures) and about the possible differences of their EPS in biofilms or biocrusts. This work aimed to characterize *P. ambiguum* and *L. ohadii* upon water deprivation in terms of the amount, monosaccharidic composition, and MW distribution of the soluble soil EPS fraction (loosely bound EPS, LB-EPS) and of the condensed soil EPS fraction (tightly bound EPS, TB-EPS) in biocrust-forming conditions, and of the RPS and anchored polysaccharides (either sheath or glycocalyx EPS) in biofilm-forming conditions. Understanding these differences could reveal the diversity of existing mechanisms to tolerate harsh environmental conditions, such as water-deprivation, determining relevant information for potential biotechnological applications.

### **2. Materials and Methods**
