Glycogen, poly(3-hydroxybutyrate) and pigment accumulation in three Synechocystis strains when exposed to a stepwise increasing salt stress

Vol.:(0123456789) 1 3 https://doi.org/10.1007/s10811-022-02693-3 Glycogen, poly(3‑hydroxybutyrate) andpigment accumulation inthree Synechocystis strains whenexposed toastepwise increasing salt stress K.Meixner1,2· C.Daffert1· D.Dalnodar1· K.Mrázová3· K.Hrubanová3· V.Krzyzanek3· J.Nebesarova4,5· O.Samek3· Z.Šedrlová6· E.Slaninova6· P.Sedláček6· S.Obruča6· I.Fritz1 Received: 21 September 2021 / Revised and accepted: 7 January 2022 © The Author(s) 2022 Abstract The cyanobacterial genus Synechocystis is of particular interest to science and industry because of its efficient phototrophic metabolism, its accumulation of the polymer poly(3-hydroxybutyrate) (PHB) and its ability to withstand or adapt to adverse growing conditions. One such condition is the increased salinity that can be caused by recycled or brackish water used in cultivation. While overall reduced growth is expected in response to salt stress, other metabolic responses relevant to the efficiency of phototrophic production of biomass or PHB (or both) have been experimentally observed in three Synechocystis strains at stepwise increasing salt concentrations. In response to recent reports on metabolic strategies to increase stress tolerance of heterotrophic and phototrophic bacteria, we focused particularly on the stress-induced response of Synechocystis strains in terms of PHB, glycogen and photoactive pigment dynamics. Of the three strains studied, the strain Synechocystis cf. salina CCALA192 proved to be the most tolerant to salt stress. In addition, this strain showed the highest PHB accumulation. All the three strains accumulated more PHB with increasing salinity, to the point where their photosystems were strongly inhibited and they could no longer produce enough energy to synthesize more PHB. Keywords Salt stress· Synechocystis sp.· Poly(3-hydroxybutyrate)· Glycogen· Pigments Introduction Cyanobacteria have a long evolutionary history in which they colonized a wide variety of habitats, ranging from glaciers to hot springs, from freshwater to saline environments. They evolved different strategies to cope with environmental stress and they manage to adapt to changing environmental conditions within their habitats. Cyanobacteria are capable of acclimating to various stress conditions, by adapting their physiological, biochemical and molecular activities. They respond to temperature stress by altering membrane composition to adapt fluidity through saturation of fatty acids and protein integrity. Strategies used in response to UV stress include scavenging reactive oxygen species, synthesis of UV-absorbing/shielding compounds, Highlights • Synechocystis sp. adapted to an otherwise lethal salinity of 6.5 %. •Pigment amounts and relations changed with increasing salinity. •In stressed cells increased PHB but decreased glycogen amounts occurred. •The highest amount of PHB accumulated in Synechocystis salina CCALA192 at 4.5 % NaCl. * K. Meixner Kathar[email protected] 1 Institute ofEnvironmental Biotechnology, Department ofAgrobiotechnology, IFA-Tulln, University ofNatural Resources andLife Sciences, Vienna, Konrad-Lorenz-Straße 20, 3430Tulln, Austria 2 BEST Bioenergy andSustainable Technologies GmbH, Inffeldgasse 21b, 8010Graz, Austria 3 Institute ofScientific Instruments, The Czech Academy ofSciences, Královopolská 147, 61264Brno, CzechRepublic 4 Institute ofParasitology, Biology Centre, The Czech Academy ofSciences, Branisovska 31, 37005CeskeBudejovice, CzechRepublic 5 Faculty ofScience, Charles University, Vinicna 7, 12844Prague2, CzechRepublic 6 Faculty ofChemistry, Brno University ofTechnology, Purkynova 118, 61200Brno, CzechRepublic / Published online: 30 March 2022 Journal of Applied Phycology (2022) 34:1227–1241 1 3 repair of damaged DNA and resynthesis of proteins. Against metal-induced stress, cyanobacteria protect themselves by altering their membrane structure and activating or inactivating transfer pumps. Besides that, salt stress is a major abiotic stress problem in arid and semi-arid regions as well as in irrigation areas (Sudhir and Murthy 2004). Salinity reduces the availability of water and increases the Na+ and Cl− concentration. Cyanobacteria reduce the Na+-uptake as well as their active efflux via Na+/H+ antiporter, they induce organic compounds, increase antioxidative defense systems to detoxify the reactive oxygen species, accumulate compatible solutes to compensate osmotic pressure and finally, also express salt-inducible proteins to cope with salt stress (Klähn and Hagemann 2011; Rezayian etal. 2019). Besides these survival strategies, cyanobacteria, such as Synechocystis sp., or heterotrophic bacteria, such as Cupriavidus necator accumulate carbon and energy storage polymers. It is known that the lipophilic poly(3-hydroxybutyrate) (polyhydroxybutyrate,PHB) is formed under nitrogen and/or phosphorus deficiency with simultaneous excess of carbon and energy. PHB is known to act in heterotrophic bacteria as a protectant against osmotic and oxidative stress, against pH fluctuations and increased pressure (Obruca etal. 2020, 2021; Fritz etal. 2021). Besides PHB, cyanobacteria produce the hydrophilic polymer glycogen, which is increasingly synthesized during the stationary phase (Velmurugan and Incharoensakdi 2018) and is stated to be rather a quick-response energy and carbon storage to cope with darkness (night), when its degradation may be the only available energy source for the strict phototrophic cells (Damrow etal. 2016; Koch and Forchhammer 2021). Furthermore, glycogen accumulation is crucial for phycobilisome degradation and assembly of the thylakoid membrane in presence of light (Velmurugan and Incharoensakdi 2018). It is not fully understood why cyanobacteria produce PHB and glycogen in parallel, as both storage compounds compete for the carbon pool (Wu etal. 2002; Rueda etal. 2020; Fritz etal. 2021). The glycogen synthesis path from CO2 starts with 3-phosphoglycerate, which can also be converted into pyruvate, then into acetyl-CoA and finally into PHB (Rueda etal. 2020). However, PHB and glycogen levels are independent of each other (Velmurugan and Incharoensakdi 2018; Koch etal. 2019). Glycogen concentrations in Synechocystis PCC6803 are stated to range from 18.5% (Velmurugan and Incharoensakdi 2018) up to 50%(per cell dry weight, CDW) (Luan etal. 2019). The concentration rises with cultivation and peaks at the mid-stationary phase (22.7%CDW), additionally it increases under nitrogen deficiency (36.8% CDW) and glucose addition (41.3% CDW) (Monshupanee and Incharoensakdi 2014). So does PHB, whose maxima are reported to be about 10–20% (CDW) in chlorotic cells and can be further increased by genetic engineering to 63% when cultivated in nitrogenand phosphorous-depleted medium and to 81% when acetate is added (Koch etal. 2020). Initial concentrations of glycogen and PHB in Synechocystis PCC6803 are reported to be approximately 12% and 1%, respectively (Dutt and Srivastava 2018). The experiments conducted herein aimed at evaluating to what extent Synechocystis sp. can survive and adapt to stepwise increasing salt concentrations and how biomass concentration changes, especially in regard to glycogen, PHB and pigments. For these purposes, growth and cell composition of three Synechocystis strains were monitored at stepwise increasing salt (NaCl) concentrations (0% to 8%). Material andMethods Strains andMedia The strains Synechocystis sp. PCC6803, Synechocystis cf. salina CCALA192 and an isolated wild-type Synechocystis strain, called IFA3, were used. PCC6803 was obtained from the Pasteur culture collection (FRA) and CCALA192 from the culture collection of autotrophic organisms (CZE). IFA3 was isolated in 2018 from a pond in Lower Austria and identified by sequencing its 16S-rDNA (see supplementary material). The strains were cultivated in a mineral medium based on BG-11 (Rippka etal. 1979), whose nitrogen and phosphorous contents were adjusted to allow an early biomass growth until starvation of nitrogen and phosphorous initializes PHB production within a single cultivation stage. Nitrogen limitation is indicated by a typical color switch from blue-green to olive-green and orange. Composition mineral medium per liter: NaNO3: 0.45g, Fe(NO3)3.9H2O: 0.025g, MgSO4.7H2O: 0.10g, CaCl2.2H2O: 0.60g, Na2CO3: 0.20g, K2HPO4: 0.08g, trace element solution 1.50mL. Composition trace element solution per liter H3BO3: 0.509g, CuSO4.5H2O: 0.150g, KI: 0.181g, FeCl3.6H2O: 0.293g, MnSO4.H2O: 0.296g, Na2MoO4.2H2O: 0.082g, NiSO4.6H2O: 0.275 g, Co(NO3)2.6H2O: 0.100 g, ZnSO4.7H2O: 0.490 g, KAl(SO4)2.12H2O: 0.395 g, KCr(SO4)2.12H2O: 0.470g. Experimental setup In the herein conducted experiments, the salt (NaCl) concentration in the medium was stepwise increased. Cultivation of each stage was carried out in 200mL mineral medium in 500-mL Erlenmeyer flasks in order to achieve a thin layer thickness of the cultures. The flasks were shaken manually two times per day. For cultivation ambient CO2 concentration (419ppm – (McGee 2021)), 22°C, 75.9 ± 9.5µmol 1228 Journal of Applied Phycology (2022) 34:1227–1241 1 3 photons m−2 s−1 (metal halide lamp, Philips Master HPI-T Plus, 250W) and a day/night cycle of 16/8h were provided. The experiments were carried out as triplicates, each cultivation stage lasted 42days. Cells were transferred from the current to the next stage between weeks two and four (see arrows in Fig.1), depending on cell density (OD750). The OD750 at which the inoculum was taken was 2.20 ± 0.37 for PCC6803, 2.36 ± 0.39 for CCALA192 and 1.98 ± 0.74 for IFA3. For the inoculation, about 40mL of culture from each flask of the current stage were harvested (pooled from triplicates) and cell numbers were counted via Thoma chamber. All of the suspension was centrifuged gently at 800×g, for 15min, the cells were resuspended in the necessary amount of salt-containing mineral medium to get a cell density of 1.2–2 × 108 mL−1. Thereof, 20mL were transferred into 180mL medium of the next step, resulting in 1.2–2 × 107 cells mL−1. The NaCl concentration of the stages was set to 0% (w/v, reference), 2% (342mM), 3% (513mM), 4% (684mM), 4.5% (770mM), 5% (856mM), 5.5% (941mM), 6% (1027mM), 6.5% (1112mM) and 8% (1369mM), respectively. Growth monitoring andBiomass analysis Evaporation was compensated by adding the necessary amount of deionized water to each flask before samples were taken. Growth was monitored via optical density at 435nm (OD435) and 750nm (OD750) twice a week, also pH was measured in those samples. Once per week, the composition of the biomass was analyzed. Therefore, cells were separated from the cultivation media and washed with reverse osmosis water by centrifugation. Aliquots of wet biomass were stored at -20°C until analysis of glycogen and pigments, and aliquots were dried at 105°C for determination of cell dry weight (CDW) and PHB. The cell number of each sample was estimated by spectral flow cytometer (Cytek Aurora) where samples were individually washed and properly diluted by phosphate buffer (50mM, pH 8). For analyzing the PHB content, the dried pellet was digested with 200 µL concentrated (98%) sulfuric acid (H2SO4) for 30min at 90°C (adapted after (Karr etal. 1983)). In this step, PHB was converted to crotonic acid. Afterwards, the samples were filled to 10mL with deionized water and prepared for HPLC (high-performance liquid chromatography) analysis (Agilent 1100; column: Transgenomic COREGEL 87H3; detector: Agilent 1100 RI). The glycogen content of the cells was determined based on Koch etal. (2019). The pellet of 2mL sample was resuspended in potassium hydroxide (KOH, 30% w/v) and incubated at 95°C for 2h. Ice cold ethanol (absolute) was added and the sample was incubated at -20°C overnight to precipitate glycogen. Subsequently, the pellet was washed twice with ethanol (1st wash: 70%, 2nd wash: absolute), dried at 60°C, resuspended in sodium acetate buffer (100mM, pH 4.5), and digested with amyloglucosidase solution (0.62 U µL−1) at 55°C and continuous shaking for 2h. Carrez precipitation (method for removing turbidity such as colloids (Gänzle 2021)) was carried out to clarify the liquid and remove the enzyme. Finally, the sample was prepared for HPLC analysis (same configuration as for PHB analysis). For analyzing contents of chlorophylla and total carotenoids (carotenoids) as well as c-phycocyanin (phycocyanin), the washed wet biomass was extracted with ethanol (absolute) (Lichtenthaler and Wellburn 1983; Ritchie 2006) and reverse osmosis water, respectively. Absorbances of the extracts were measured with a UV–Vis spectrometer (Shimadzu UV-1800). Pigment concentrations were calculated from the absorbance values of the extracts at certain wavelengths (chlorophylla: 665 nm and 750 nm (Ritchie 2006); total carotenoids: 470 nm and 750 nm, modified after Lichtenthaler and Wellburn (1983); c-phycocyanin: 615 nm and 652 nm (Bennett and Bogorad 1973)) with equations (1-3): Fig. 1 Time curve of the conducted experiments. Cells from the current stage were transferred to the next stage between weeks two and four (arrows) 1229Journal of Applied Phycology (2022) 34:1227–1241 1 3 Extraction-based pigment analysis was supplemented by their spectroscopic detection directly in cyanobacterial cell suspensions using diffusive transmission spectrophotometry. For this purpose, undiluted samples were measured in silica cuvettes on UV–Vis spectrophotometer Hitachi U-3900H with integrating sphere 60mm DIA at scan speed 600nm min−1. Ultrastructure For ultrastructural analysis, cyanobacterial cultures were centrifuged (4min, 1000×g). Samples for cryogenic scanning electron microscopy (cryo-SEM) were prepared by pipetting the concentrated pellet of cells on 6-mm Al carrier type A and closing with the flat side of type B. Samples were fixed using the high-pressure freezing method (EM ICE, Leica Microsystems) without using any cryo-protectant. Frozen samples were transferred into a cryo-vacuum preparation chamber (ACE600, Leica Microsystems) and then underwent freeze fracturing and sublimation at -95°C for 7min. No metal coating was applied. Following sublimation, cyanobacterial cells were observed in a scanning electron microscope (Magellan 400/L, FEI) equipped with a cryo stage at -120°C using a 1–2keV electron beam. For transmission electron microscopy (TEM), the concentrated pellet of cells was pipetted on 3-mm Al carriers covered with 1% solution of lecithin in chloroform and fixed using the high-pressure freezing method (EM ICE, Leica Microsystems). Frozen samples were then transferred into a freeze-substitution unit (AFS2, Leica Microsystems). Substitution solution contained 1.5% OsO4 in acetone, the protocol used for the freeze-substitution was previously described in Kouřilová etal. (2021). After freeze substitution, samples were washed three times in acetone for 15min each and gradually infiltrated with medium-hardness epoxy resin (Epoxy embedding medium, Sigma-Aldrich). Infiltration mixtures of epoxy resin and acetone in ratios 1:2, 1:1, 2:1, 1:0 were changed after 1h. After the final exchange, samples were left in fresh pure resin under a vacuum in a desiccator overnight. Samples in fresh resin were cured using 62°C heat for 48h and cut to ultrathin sections on ultramicrotome (Ultracut UCT, Leica Microsystems) using a diamond knife (Diatome) with 45° cutting angle. Sections on 300 mesh (1) Chlorophylla[ mg L− 1] =11.90355 ∗ ( A665 −A750 ) (2) Total carotenoids [mg L−1]= 1000 ∗ ( A470 −A750 ) −2.05 ∗chlorophylla [ mg L− 1] 245 (3) C −phycocyanin[mg L−1]= A 615 − 0.474 ∗ A 652 5.34 ∗1000 copper grids were then stained using solutions of uranyl acetate and lead citrate and finally observed in transmission electron microscope JEOL 1010 using accelerating voltage 80kV and images were digitally recorded by CCD camera Megaview III (Olympus). Results andDiscussion Three Synechocystis strains (PCC6803, CCALA192, IFA3) were exposed to stepwise increasing salt concentrations to evaluate how far they are able to adapt to increasing salt concentrations (from 0 to 8% w/v NaCl) and how biomass composition changes. The growth of CCALA192 (OD750) in all salt concentrations is shown as an example in Fig.2 (those of PCC6803 and IFA3 are in the supplementary material). Salt concentrations of 0%, 3%, 6% and 8% (shown as solid lines) were chosen to illustrate the effect on growth and biomass composition (Figs.3, 4, and 5). These stages represent no (0%, reference), moderate and high salt concentrations. The intermediate salt concentrations were necessary to gradually adapt the cells to the increasing salinity. The results of these stages do not show outstanding values nor do they provide different insights. Therefore, we refrained from presenting all data in the main section of this manuscript. The whole data (growth curves at OD750, OD485 [-], OD750[-], pH [-], CDW [g L−1], PHB [mg L−1], glycogen [mg L−1], chlorophylla [mg L−1], phycocyanin [mg L−1], total carotenoids [mg L−1]) can be found in the supplementary material. Growth The cell dry weight (CDW) of all the three Synechocystis strains (PCC6803: Fig.3a, CCALA192: Fig.4a, IFA3: Fig.5a) was highest in the reference cultivations, without salt addition (0% NaCl). CCALA192 demonstrated the highest biomass concentrations (2.08 ± 0.04g L−1) followed by IFA3 (1.98 ± 0.07g L−1) and PCC6803 (1.85 ± 0.03g L−1). For all three strains, it is clear that growth decreased with increasing salt concentrations and stagnated at a salt concentration of 8%. In the medium with 3% salt the final biomass concentrations of all three strains were comparable – PCC6803: 1.54 ± 0.02 g L−1, CCALA: 1.55 ± 0.08 g L−1, IFA3: 1.41 ± 0.09g L−1. In the medium with 6% salt IFA3 showed the highest biomass concentrations (1.11 ± 0.05g L−1), followed by CCALA192 (1.06 ± 0.09g L−1) and PCC6803 (0.90 ± 0.01g L−1). In 8% NaCl only IFA3 showed slight growth and reached 0.39 ± 0.03g L−1 after a cultivation time of 42days while both, CCALA192 and PCC6803, did not show any growth. Trends in cell numbers of cultures of CCALA192 and PCC6803 (42days of cultivation), 1230 Journal of Applied Phycology (2022) 34:1227–1241 1 3 determined by flow cytometry (data not shown) and CDW, respectively, corresponded to each other. As expected, the exposition of cyanobacterial cultures to stepwise increasing salinities in cultivation media partially inhibited the growth of the cells, with the inhibitory effect being more pronounced the higher the salt concentration in the culture medium. Velmurugan and Incharoensakdi (2018) stated that growth of moderately halotolerant cyanobacteria Synechocystis is hardly affected up to 100mM (0.6%) NaCl and results in a biomass concentration of 1.4g L−1. With increasing salt concentrations, biomass concentrations declined from 1.3g L−1 (200mM = 1.2% NaCl) to 1.1g L−1 (300mM = 1.8% NaCl) (day 20). These values (achieved at 100µmol photons m−2 s−1, 28°C, in BG-11 medium) are within the range of those achieved by our experiments (0.61 to 0.96g L−1 at 2%NaCl and day 21, 1.55 to 2.04g L−1 at 2% NaCl and day 42). Also, achieved results about biomass were confirmed by data from flow cytometer, i.e., cultures with increasing salt concentrations demonstrated decreasing trend also in cell number (data not shown). PHB All three strains produced PHB. The cellular concentration of PHB increased over cultivation time (PCC6803: Fig.3b, CCALA192: Fig.4b, IFA3: Fig.5b). In the reference cultivations (0% NaCl), lowest PHB concentrations were achieved. The PHB content increased up to a certain salt concentration, which was individually different for each strain and was then decreasing when salinity further rose. The highest PHB concentration (6.98 ± 0.10% CDW) was achieved by CCALA192 in 4% salt after 42days of cultivation, followed by PCC6803 (3.60 ± 0.28% CDW) in 3% and IFA3 (3.27 ± 0.8% CDW) in 2%, after 42 and 36days, respectively. The same trend is visible when comparing PHB concentrations of the strains after the same cultivation time (42days) in 3% salt—CCALA192: 4.43 ± 0.10% CDW, PCC8603: 3.60 ± 0.28% CDW, IFA3: 2.33 ± 0.19%CDW. At all the salt concentrations, CCALA192 accumulated higher PHB amounts than the other two strains. Fig. 2 Growth curves of all experiments conducted with Synechocystis CCALA192. The solid lines show salt concentrations of 0%, 3%, 6% and 8% and are described in more detail in the text. The arrows indicate the inoculation of the next stage. n = 3 1231Journal of Applied Phycology (2022) 34:1227–1241 1 3 The PHB contents of all the three strains in the reference cultivations (0% NaCl) were by far lower (0.21 to 0.61% CDW, day 21) than reported for Synechocystis PCC6803 (about 2.5% CDW, day 20, at 100µmol photons m−2 s−1, 28°C, in BG-11 medium) (Velmurugan and Incharoensakdi 2018) and for nitrogen depleted cells (about 15–16.5% CDW, day 12, at 150µmol photons m−2 s−1, 30°C, in BG-11 medium + 0.4%(v/v) acetate) (Dutt and Srivastava 2018). PHB concentration increased with increasing cultivation time but did not exceed 2% CDWin PCC6803, when cultivation was stopped on day 42. Reasons could be the lower temperature and light intensity in our experiments lead to lower growth compared to literature. And most likely that nitrogen deprivation occurred later than day 20 or 12, due to the used medium, which we adapted for single-stage cultivation. Furthermore, no organic carbon was added as described by Dutt and Srivastava (2018). IFA3 and CCALA192 contained 2.1% and 2.7% CDW at the end of cultivation. Glycogen Glycogen concentrations (PCC6803: Fig.3b, CCALA192: Fig.4b, IFA3: Fig.5b) were—comparable to PHB concentrations—increasing in all the three strains with increasing cultivation time. Another similarity to PHB accumulation was that CCALA192 also had the highest glycogen concentrations but in contrast in 3% NaCl. The second highest glycogen concentration was achieved by PCC6803, followed by IFA3. In all the strains the highest glycogen levels were accumulated in 3% NaCl—CCALA192 (49.43 ± 0.58% CDW) after 42, by PCC6803 (48.60 ± 1.82% CDW) and IFA3 (47.24 ± 0.73% CDW) after 35days. With further increasing salt concentrations glycogen levels decreased. This effect is clearly visible later in cultivation (> day 29). The glycogen concentrations in the reference cultivations (0% NaCl) obtained 12.45 ± 0.66%CDW (PCC6803), 23.04 ± 1.21% CDW (CCALA192) and 12.80 ± 0.48% CDW (IFA3) on day 21. By increasing salinity to 6%, glycogen concentrations of 20.67 ± 1.25% CDW (PCC6803), 20.93 ± 0.63% CDW (CCALA192) and 20.26 ± 1.48% CDW (IFA3) were achieved on day 21, while on day 42 it had just increased toward 27.24 ± 1.18% CDW, 33.46 ± 2.63% CDW and 35.05 ± 1.66% CDW in PCC6803, CCALA192 and IFA3, respectively. In contrast to PHB, glycogen levels were high at low salt concentrations. PHB and glycogen in common increased Fig. 3 PCC6803 (a) growth, (b) PHB (filled columns) and glycogen (dashed columns), (c) chlorophylla (filled columns) and phycocyanin (dashed columns) as well as (d) carotenoids in 0%, 3%, 6% and 8% NaCl added to the medium; n = 3, error bars = standard deviation 1232 Journal of Applied Phycology (2022) 34:1227–1241 1 3 up to certain salt concentrations (e.g., 3% NaCl for glycogen and 4% NaCl for PHB, both in CCALA192) and then declined. For glycogen, higher concentrations are stated to be produced in 4% NaCl than in 0% (Pade etal. 2017). When comparing reference cultivations (0% NaCl) of Synechocystis PCC6803 higher glycogen values were achieved in the literature—24.1% CDW within 21days (Velmurugan and Incharoensakdi 2018) —than compared to herein cultivated PCC6803 and IFA3 (12.45 ± 0.66%CDW and 12.80 ± 0.48% CDW, respectively, in reference cultivations (0% NaCl), day 21). The values obtained by CCALA192 (23.04 ± 1.21% CDW, reference cultivations (0% NaCl), day 21) were within the range. By genetic modifications (glycogen overexpression combined with phaA-knockout), glycogen concentrations were further increased to 38.8% CDWon day 20 and 40.4% CDWon day 25 (Velmurugan and Incharoensakdi 2018). These values were twice as high as those achieved in the reference cultivations (0% NaCl) on day 21 but within the range obtained at 3% NaCl after 42 cultivation days (PCC6803: 45.84 ± 2.02% CDW; CCALA192: 49.43 ± 0.58% CDW; IFA3: 42.85 ± 1.08% CDW). Glycogen concentrations can be even further enhanced by nitrogen deprivation (to about 42% CDW, after 12days). Besides that, glycogen concentrations especially at the beginning of cultivation are elevated when cells are pre-grown under photomixotrophic conditions (slightly below 60% CDW at the beginning of cultivation and slightly above 50% CDW after 12days) compared to phototrophic conditions (about 3% CDW at the beginning and slightly over 40% CDW after 12days) (Dutt and Srivastava 2018). Salt Stress andits Effect ontheAccumulation ofPHB andGlycogen The average PHB-to-glycogen ratios over the cultivation time in 3% salt were comparable between all three strains (1:21 for CCALA192, 1:24 for PCC6803 and 1:27 for IFA3), suggesting that PHB and glycogen synthesis pathways in all three strains were about equally strong at moderate salt concentrations. With increasing or decreasing salt concentrations this ratio changed. PCC6803 produced 40 and 147 times more and IFA3 55 and 129 times more glycogen than PHB at 0% and 6% NaCl, respectively. Only glycogen accumulation in CCALA192 stood rather constant at 1:25 and 1:29 in 0% and 6% NaCl. Summarized, all three strains accumulated more glycogen than PHB, but ratios changed with changing salinity. The fact that medium Fig. 4 CCALA192 (a) growth, (b) PHB (filled columns) and glycogen (dashed columns), (c) chlorophylla (filled columns) and phycocyanin (dashed columns) as well as (d) carotenoids in 0%, 3%, 6% and 8% NaCl added to the medium; n = 3, error bars = standard deviation 1233Journal of Applied Phycology (2022) 34:1227–1241 1 3 salinity substantially affects PHB and glycogen content in cyanobacteria is not only of fundamental interest but it might be also of biotechnological importance since NaCl addition could be used as a tool to enhance PHB synthesis; nevertheless, the salt should be applied at later stages of cultivation to not inhibit the growth of the culture (since PHB is an intracellular metabolite and high CDW values are the necessary prerequisite for high PHB titers). The fact that fractions of both storage compounds are lower in cultures exposed to high osmotic pressures can be attributed to inhibition of thephotosynthetic system resulting in a decreased amount of carbon and energy for storage polymers synthesis (see further in the text). Nevertheless, it is also likely that carbon and energy expenses of stressresponse against osmotic pressure (excretion of Na+, production of compatible solutes, modulation of gene expression etc.) also contributed to decreased amounts of storage polymers in the cultures. Hence, if NaCl should be considered as a tool to improve PHB production, its concentration must be wisely chosen and precisely controlled since above acertain valueit has a negative impact on PHB synthesis. Spectroscopic Identification ofPigments inCell Suspensionsof Synechocystis strains PCC6803 andCCALA192 To identify and analyze pigments directly in the cultivated culture, we used a less common spectroscopic method of diffusive transmission spectrophotometry that allows the determination of UV-VIS absorption spectra for highly turbid samples. All the measured cultures were at the same stage of cultivation (42days). As can be seen from the comparison of corresponding pigment contents shown in Fig.3c/d, Fig.4c/d, Fig.5c/d and changes in the spectral fingerprints of the pigments in Fig.6, there is a good agreement in the observed effect of the salt present in cultivation media on the changes in pigments composition. Quantification ofPigmentsobtained byExtraction Chlorophylla In all the three Synechocystis strains, the chlorophylla contents rose until day 14 (0% and 3% NaCl, PCC6803, CCALA192, IFA3) or 21 (6% NaCl, PCC6803, CCALA192, Fig. 5 IFA3 (a) growth, (b) PHB (filled columns) and glycogen (dashed columns), (c) chlorophylla (filled columns) and phycocyanin (dashed columns) as well as (d) carotenoids in 0%, 3%, 6% and 8% NaCl added to the medium; n = 3, error bars = standard deviation 1234 Journal of Applied Phycology (2022) 34:1227–1241 1 3 IFA3) and then decreased with increasing cultivation time. Additionally, chlorophylla tends to decrease at higher salt stress levels (PCC6803: Fig.3c, CCALA192: Fig.4c, IFA3: Fig.5c). Chlorophylla was highest in PCC6803 (1.34 ± 0.02% CDW) in 2% NaCl on day 14, compared to IFA3 and CCALA192, whose chlorophylla levels also peaked (1.01 ± 0.03% CDW and 0.93 ± 0.06% CDW, respectively) in 2% NaCl on day 14. Concerning cultivation time, an increasing trend of chloropylla in Synechocystis PCC6803 and engineered strains (glycogen overexpression, phaA-knockout) thereof is reported for growth in salt free BG-11 medium from day 10 to 15, followed by a decrease to day 20 (Velmurugan and Incharoensakdi 2018). This trend could be confirmed by PCC6803, CCALA192 and IFA3 whose chlorophylla content in reference cultivations (0% NaCl) rose until day 14 or 21 and then declined until the end of cultivation. The results obtained for chlorophylla at different salt concentrations were comparable with those collected by Sudhir and Murthy (2004), who stated that at 342mM (2%) NaCl the chlorophylla content in Synechocystis PCC6803 increased, while at 684mM (4%) or 1026mM (6%) it sharply decreased. As the chlorophylla content decreased, growth also decreased but did not stop completely. The decrease in chlorophyllawas not only observed with cultivation time, but also with increasing salt concentration. Higher salt concentrations led to a faster decline in chlorophylla. However, even low chlorophylla concentrations were sufficient for Synechocystis to continue growing. In case of nitrogen chlorosis, phycocyanin (peak at 630nm) and to a lesser extent chlorophylla (peak at 680nm) gradually decreased with increasing cultivation time (Krasikov etal. 2012) and growth stopped (Klotz etal. 2016). In glycogen overexpressing strains, chloropylla contents were lower compared to the wild type and phaA-knockout strain, respectively. Despite low chlorophylla contents, oxygen evolution rate was highest in the combined strain (glycogen overexpression and phaA-knockout), which enhanced overall biomass concentration (Velmurugan and Incharoensakdi 2018). Similar patterns can be seen when comparing chlorophylla and glycogen contents of PCC6803 and CCALA192. Chlorophylla contents of PCC6803 were generally higher, while glycogen levels and growth were slightly lower than in CCALA192. This suggests that the efficiency or the content of PSII (photosystem II,to which oxygen evolution is connected (Allakhverdiev and Murata 2008)) in PCC6803 is rather low compared to the other two strains. Phycocyanin Phycocyanin showed similar tendencies as chloropylla; its contents rather decreased with increasing cultivation time (PCC6803: from day 7 at 3%, from day 29 at 6%; CCALA192: from day 7 at 0%, from day 14 at 3%, from day 21 at 6%; IFA3: from day 7 at 0% and 3%, from day 21 at 6%) (PCC6803: Fig.3c, CCALA192: Fig.4c, IFA3: Fig.5c). Related to salt concentrations, the picture is more complex: The highest phycocyanin levels were reached by CCALA192 (9.17 ± 1.38% CDW) in 3% NaCl at day 14 and by IFA3 (7.17% CDW) in 0% at day 7. PCC6803 reached a maximum of only 2.34 ± 0.05% CDW in 3% NaCl on day 7. In 4% NaCl phycocyanin concentrations were comparably low—the maximum was 2.32 ± 0.11% CDW in CCALA192 at day 14. At higher salinities, phycocyanin Fig. 6 VIS absorption spectra (400–750nm) of cultures after 42days of cultivation in 0%, 3%, 6% and 8% NaCl added into the medium a) PCC6803 b) CCALA192. Spectra were obtained by diffusive transmission spectrophotometry 1235Journal of Applied Phycology (2022) 34:1227–1241