*3.3. Effects of Water Deprivation in Chlorophyll a and EPS Production in Biofilms*

For the controls and the water-deprived biofilms, no cyanobacterium produced significantly more chlorophyll *a* than the other (Figure 4). Therefore, no significant correlation could be attributed between the EPSs contents and chlorophyll a (*p* > 0.05).

For both cyanobacteria, the amount of RPS and sheath or G-EPS did not show significant differences between the control and the water-deprived biofilms (Figure 5A,B). For *P. ambiguum*, the sheath EPS amount was significantly higher, under all conditions, than the RPS, indicating that the sheath gives the major contribution to the total carbohydrates of this cyanobacterium (Figures 5A and S4B) in biofilm forming conditions. For *L. ohadii* under water deprivation stress, the G-EPS was significantly higher than the RPS, but it was not significantly higher than the control RPS (Figure 5B). Moreover, when comparing the RPS between the cyanobacteria in each condition, *L. ohadii* RPS after water deprivation stress resulted significantly higher (Figure S5A). On the other hand, the sheath EPS resulted significantly higher than G-EPS in water deprivation stress, indicating how sheath EPS could contribute to *P. ambiguum* as protection in biofilms (Figure S5B).

#### *3.4. Changes in Monosaccharidic Composition and Molecular Weight Distribution of Biofilms EPSs*

The monosaccharidic composition revealed differences in biofilm-cultures, showing different profiles between the strains, the conditions, and comparing them to the biocrusts. The RPS after water deprivation were mainly composed of glucose and uronic acids (Figure 6A,B). After water deprivation, only glucuronic acid demonstrated significantly higher molar ratio than the control for *P. ambiguum* RPS. Unlike *P. ambiguum* LB-EPS, the molar ratio of glucose in *P. ambiguum* RPS did not show significant differences between the control and water deprivation. The RPS after water deprivation showed higher equitability than the RPSs extracted from the control biofilms, conferring the highest diversity, despite the smaller number of monosaccharides (Table S2). The sheath EPS after water deprivation showed lower diversity than the RPS after water deprivation and the sheath EPS control, due to the higher dominance of glucose. This moiety also demonstrated significantly higher molar ratio than the control in the sheath EPS (Figure 6C, Table S2).

For all the conditions, the RPSs demonstrated higher diversity than the G-EPS in *L. ohadii* biofilm (Table S2). For this cyanobacterium RPS, the galacturonic and glucuronic acids were significantly higher in water deprivation compared to the control. A similar profile was found in *P. ambiguum* biofilm, in which these sugars dominated the RPS after water deprivation. Unlike *L. ohadii* LB-EPS, the molar ratio of glucose in *L. ohadii* RPS after water deprivation was significantly lower compared to the control. In addition, unlike *P. ambiguum* biofilm, the *L. ohadii* RPS in water deprivation showed lower diversity compared with the RPS control. The latter resulted in the highest number of monosaccharides and the highest equitability, leading to the highest diversity among all the *L. ohadii* EPS fractions. Finally, glucose and galactose were the monosaccharides mostly present in G-EPSs (Figure 6D). The G-EPS after water deprivation showed lower diversity than the RPS after water deprivation and the G-EPS control, due to the higher dominance of glucose as observed in the sheath EPS after water deprivation (Figure 6B–D, Table S2).

**Figure 6.** Monosaccharidic composition of the EPS extracted from biofilms: (**A**) RPS from *P. ambiguum*, (**B**) RPS from *L. ohadii*, (**C**) Sheath EPS, (**D**) G-EPS. Molar percentages (%) of single sugars are represented (expressed as moles of the single monosaccharide divided by the total amount of moles of monosaccharides in the EPS × 100). Symbol \*, when present, indicates significant differences between the control and the water-deprived period in each monosaccharide. Fuc, fucose; Rha, rhamnose; GalN, galactosamine; Ara, arabinose; GlcN, glucosamine; Gal, galactose; Glc, glucose; Man, mannose; Xyl, xylose; Fru, fructose; Rib, ribose; GalA, galacturonic acid; GlcA, glucuronic acid.

The EPS extracted from biofilms were analyzed in terms of molecular weight (MW) distribution. The MW distribution of the RPSs extracted were not detectable and data are not shown. The sheath EPSs of *P. ambiguum* were predominantly composed of molecules lower than 50 kDa, while the *L. ohadii* G-EPSs were constituted of molecules having an apparent MW between 410 kDa and 50 kDa (data not shown).

#### **4. Discussion**

Filamentous cyanobacteria have been studied for their potential to survive water deprivation [29,44,45]. Previous studies demonstrated that the profile of exopolysaccharides is heterogeneously dependent on the strain [17,26,42]. In this study, two different strains were cultivated as biocrusts and biofilms, showing different responses to water deprivation stress.

#### *4.1. EPS and Growth Profile in Biocrust-Forming Cyanobacteria*

When the two cyanobacteria were incubated in microcosms, the amount of chlorophyll *a* decreased under water deprivation, indicating that five days deprivation of water had an impact in photosynthesis. The photosynthetic apparatus has been reported to respond to water deprivation conditions [30,31,46–48]. These authors reported that the phycobilisomes in the desiccated state lose their organized structure, and after re-wetting, photosynthetic activity was reactivated. Previous study identified genes encoding proteins involved in photosynthesis and chlorophyll *a* synthesis whose transcription was induced by re-wetting the biocrust of *Microcoleus vaginatus* [49]. Additionally, previous work reported that the dry phase induces an osmotic stress, which regulates genes and decreases the photosynthesis activity. Therefore, this agrees with our observation of a decrease in chlorophyll *a* induced by water deprivation [50,51].

Cultures of bacteria and cyanobacteria have been reported to increase EPS production under water deprived conditions, suggesting that resources were allocated to EPS production in response to the stress [52–54]. This may explain the apparent contradiction between the increase in EPS and the decrease in photosynthesis. In this work, the amount of the EPS changed with the water availability: *P. ambiguum* TB-EPS amount was significantly higher only under water deprivation. It was previously reported that this cyanobacterium promotes higher increase in TB-EPS compared to other cyanobacteria (i.e., *Scytonema javanicum*) [26]. In the present work, it also promoted significantly higher TB-EPS amounts compared to *L. ohadii* biocrusts under water deprivation (Figure S3B), corroborating with previously reports that *P. ambiguum* has a more prominent role of aggregation of soil particles and improvement of soil strength, due to the synthesis of large amounts of TB-EPS [25,26,36]. This could be an advantage for this cyanobacterium to deal with periods of limited water availability. The significant increase in the amount of LB-EPS of *L. ohadii* under the water deprivation stress condition corroborates previous findings [17] that the amount of LB-EPS in *L. ohadii* biocrusts increases when incubated with lower amounts of water. This suggests that the amount of water influences the amount of LB-EPS for this cyanobacterium. On the other hand, in contrast to previous reports that the amount of *L. ohadii* TB-EPS was not influenced by water availability, in the present study the amount of TB-EPS was higher under water deprivation condition, being significantly higher than the other EPSs fractions quantified for *L. ohadii* biocrust justifying their large presence in biocrusts that are subject to frequent dry and wet cycles [55].

Not only the amount of EPSs can be modulated by the harsh environmental conditions to which cyanobacteria were submitted, but also their monosaccharidic composition [8]. The higher relative abundance of glucose in the LB-EPS and TB-EPS of both cyanobacterial biocrusts, and the increase in the molar ratio of this sugar in water deprivation conditions is in agreement with previous studies that reported the higher abundance of glucose in sand biocrusts of these cyanobacteria under low water additions, mainly in LB-EPS [17,25,26]. Moreover, galactose was another sugar found in biocrusts that present higher molar ratios compared to other sugars. It has been reported to be highly produced by *P. ambiguum* TB-EPS in sand soils [25], confirming the observations on *P. ambiguum* biocrusts reported here. These data corroborate previous findings that, in nutrient limited conditions often found in biocrusts from drylands, more essential monosaccharides are required by the cyanobacteria [56], which could explain the increase in the dominance index in water deprivation stress condition.

Despite the dominance of glucose, cyanobacterial EPS are also characterized by the presence of uronic acids and deoxysugars, such as fucose and rhamnose [57–59]. In this work, uronic acids were present in significantly higher percentage after the watered periods and decreased after the water deprivation period. The uronic acids have hydrophilic character involved in the chelation of minerals, nutrients, and water molecules when present [18] and have been detected mostly under the highest water availability in *L. ohadii* LB-EPS [17]. On the contrary, fucose and rhamnose have hydrophobic character increasing cell capacity to adhere to solid surfaces [5,19]. Though the mol% of these monosaccharides is probably too low, in our results, to observe a significant difference in polymer hydrophobicity, these data still show the plasticity of the cells to modify EPSs to respond to the surrounding environment performing with the amphiphilic character of the macromolecules. In addition, the sugar amount and composition were different between the strains and EPS fractions, showing how these polymers are heterogeneously dependent on the cyanobacteria and abiotic factors.

The molecular weight distribution of EPS is another feature that deserves further investigations about its response to water deprivation. In this work, the TB-EPSs extracted were mainly composed of molecules ranging between 2 MDa–410 kDa of apparent MW. The biocrust stability was previously reported to be provided by the TB-EPS, which was composed of MW in the range 410 kDa–50 kDa, but by higher abundance of MW in the range 2 MDa–1.1 MDa [25,26,36]. Moreover, the LB-EPSs were also represented by >2 MDa–1.1 MDa molecules after water deprivation, while after the watered periods molecules in the range 1.1 MDa–410 kDa were present. The presence of high MW molecules could also be due to molecule aggregation after the extraction. However, the analysis confirms the presence of large polymers, which can contribute to the viscosity of the EPS. The presence of such high MW molecules under low moisture could indicate a higher water retention during periods of drought [17,25,60].
