Archives of Microbiology (2022) 204:165 https://doi.org/10.1007/s00203-022-02772-6 ORIGINAL PAPER Acclimation response and ability of growth and photosynthesis of terrestrial cyanobacterium Cylindrospermum sp. strain FS 64 under combined environmental factors Nadia Bahavar1 · Shadman Shokravi2 Received: 21 October 2021 / Revised: 12 January 2022 / Accepted: 14 January 2022 © The Author(s) 2022 Abstract This investigation tested the hypothesis that the native cyanobacteria can acclimatize and grow under the combination of environmental factors and/or how does their process change with the age of culture? Here, we tried to combine multiple fac- tors to simulated what happens in natural ecosystems. We analyzed the physiological response of terrestrial cyanobacterium, Cylindrospermum sp. FS 64 under combination effect of different salinity (17, 80, and 160 mM) and alkaline pHs (9 and 11) at extremely limited carbon dioxide concentration (no aeration) up to 96 h. Our evidence showed that growth, biomass, pho- tosystem II, and phycobilisome activity significantly increased under 80 mM salinity and pH 11. In addition, this combined condition led to a significant increase in maximum light-saturated photosynthesis activity and photosynthetic efficiency. While phycobilisomes and photosystem activity decreased by increasing salinity (160 mM) which caused decreased growth rates after 96 h. The single-cell study (CLMS microscopy) which illustrated the physiological state of the individual and active-cell confirmed the efficiency and effectiveness of both photosystems and phycobilisome under the combined effect of 80 mM salinity and pH 11. Keywords Alkalinity · Cyanobacteria · Cylindrospermum sp. FS 64 · Salinity · Time Abbreviations Introduction APC Allophycocyanin Chl a Chlorophyll a Cyanobacteria are a self-sufficient system that is widely CCM Carbon dioxide concentrating mechanism distributed across terrestrial and aquatic environments; and DIC Dissolved inorganic carbon terrestrial cyanobacteria plays a fundamental role in the PBS Phycobilisome biological cycle of agriculture (Mareš et al. 2014; Shokravi PSI, PSII Photosystems I and II and Bahavar 2021a). They produce various bio-available CLSM Confocal laser scanning microscopy elements such as Nitrogen and phosphorus, which are the essential nutrients for plant cultivation (Chittora et al. 2020). Moreover, they generally exhibit a high level of adaptive abilities and tolerance to a large number of environmental factor (Singh 2018). In nature, cyanobacteria are exposed Communicated by Erko Stackebrandt. to a constantly changing environment including irradiance, temperature, pH, nutrient availability, salinity, dissolved Nadia Bahavar and Shadman Shokravi contributed equally, listed inorganic carbon fluctuations (Chris et al. 2006; Bouazzara alphabetically. et al. 2020). These changes continuously expose the cyano- * Nadia Bahavar bacteria cells to multiple stressors of varying magnitude and nbahavar@gmail.com; nadia.bahavar@e-campus.uab.cat duration (Borowitzka 2018). In practice, the survival and 1 growth of cyanobacteria depend on their ability to acclimate Plant Physiology Laboratory, Bioscience Faculty, Universidad Autónoma de Barcelona, 08193 Bellaterra, varying environmental conditions. Spain Among all cultural parameters, pH is one of the most 2 Department of Biology, Gorgan Branch, Islamic Azad important factors determining cyanobacteria growth and University, Gorgan, Iran physiology (Pawlik-Skowrońska et al. 1997; Hinners et al. 13 Vol.:(0123456789) 165 Page 2 of 10 Archives of Microbiology (2022) 204:165 2015). Most cyanobacteria have the ability to grow over a So far, most studies on this genus have been focused on wide range of alkaline pH in laboratory (Kaushik 1994). molecular biology (Srivastava et al. 2009; Katoch et al. Nearly nothing is known about the mechanisms of cellu- 2016), physiological characteristics (Briand et al. 2004), lar survival, cell stability, or growth of cyanobacteria under proline accumulation (Chris et al. 2006), heavy metal stress extreme alkaline conditions (Jangir et al. 2021). Elevated pH (Singh et al. 1989; Chris 2012), Nitrogen forms effect (Ken- (alkalinity) directly influences growth rate and cell yield (de esi et al. 2009) and chemical analysis (Mareš et al. 2014). Souza Santos et al. 2011; Shokravi and Bahavar 2021a, b), The aim of this research is to characterize the acclimation enzyme activity (Li et al. 2013), biosorption (El-Din 2017), behaviors of Cylindrospermum sp. FS 64 under different pH, resistance to oxidative stress (Summerfield et al. 2013), and salinity conditions under extremely limited carbon dioxide protection (Pathak et al. 2018). It also strongly affects the concentration for 96 h. cyanobacterial abundance (Krausfeldt et al. 2019; Nguyen and Rittmann 2015). While in some cyanobacteria, the growth decreased with the increasing alkalinity (Shokravi Materials and methods and Bahavar 2021a). Therefore, evaluation the effects of dif- ferent pHs on cyanobacteria is important. In paddy fields, Culture maintenance and growth conditions the pH of floodwater varies during the day. Likewise, DIC concentration in the floodwater varies daily and seasonally Cylindrospermum sp. FS 64 was isolated from paddy fields depending on photosynthetic and respiratory rate (Pedersen of the North of Iran (Siahbalaei et al. 2011) and collected et al. 2013). The chemical equilibrium between photosyn- again by the authors in 2018. The soil samples was serially thesis and respiration implies a balance between inorganic diluted in sterilized liquid Nitrogen-free medium (BG-110) carbon and net ecosystem production (Khan et al. 2020). (Stanier et al. 1971). Isolation was done by streaking and Salinity is another environmental factor that could poten- spreading technique on solid BG-110 medium. Purification tially determine the cyanobacteria community in natural was done by alternative sub-culturing between liquid and ecosystems. Salinity as an essential factor induces diverse solid BG-110 medium (Shokravi and Bahavar 2021b). The alterations in the growth and photosynthesis (Bemal and sample was identified and described using multidisciplinary Anil 2018), biochemical like carbohydrate content (Singh approaches (Molecular 16S rRNA, and morphology using et al. 2015), and physiological characteristics of cyanobac- light, fluorescence, and phase-contrast microscopy). Strain teria (Miriam et al. 2017; Lee et al. 2021). Time (age of after identification as Cylindrospermum sp. FS 64 was coded the culture) is another essential factor in the resistance and and preserved in the algae museum of the institute of applied growth in different conditions (Alcorta et al. 2019) which sciences of Shahid Beheshti University, Tehran-Iran. The less has been considered (Bouazzara et al. 2020; Jangir et al. axenic culture were maintained in a liquid BG-110 at tem- 2021). Exposure to initial hours of new condition may create perature 30 ± 2 °C under a constant irradiance of 60 μmol a significant effect on physiological activities during the next quanta ­m−2 ­s−1 (Poza-Carrión et al. 2001). The pH was hours (Abbasi et al. 2019; Shokravi and Bahavar 2021a). adjusted in 7.8 by NaOH. However, there is increasing evidence that the combined environmental factors can be modulated by other factors Growth conditions and analysis and led to regulation, acclimation, and adaptation (Müller et al. 1993; Shokravi and Bahavar 2021a). Therefore, study- Growth of Cylindrospermum sp. FS 64—in an exponential ing environmental fluctuations in the short-time regime on growth phase—was carried out at various salinity concentra- cyanobacteria is essential to serve the sustainable develop- tions 17 (culture media without NaCl), 80 and 160 mM at ment economy in the future. alkaline pHs (9 and 11). Culture media were buffered with In the present study, we have selected the filamentous 10 mM BTP (Bis–Tris Propane) for pH 9 and 11 adjusted cyanobacterium Cylindrospermum sp. for the abundance, to the desired pH with KOH (Shokravi and Soltani 2011). fixed Nitrogen, and environmental stability. Cylindros- We studied cultures without C ­ O2 or O ­ 2 bubbling and stir- permum sp. FS 64 was isolated from paddy fields of the ring (standing condition, extremely DIC limitation) (Poza- North of Iran which description is following: aggregations Carrión et al. 2001; Shokravi and Bahavar 2021a). The bright blue-green, green–brown, sticky colonies, not regu- determination of the growth was performed using time- lar, expanded; mucilaginous, attached to margin; filaments course measurements by the correlation between optical straight, curved entangled; trichrome 9µ broad, constricted density (OD 750 nm), in vivo fluorescence, and counting at cross wall; cells variable in size, cylindrical or nearly cells according to Briand et al. (2004) using the CLMS at quadrate; heterocyst ovate or ellipsoid, terminal at one side; different salinity and alkaline pHs up to 96 h. The OD was spore oval, ellipsoidal, adjoining the heterocyst, granular, measured using Synergy HTX (Multi-Mode Microplate 10.4µ long. Reader, USA). Growth rates (µ) were calculated according 13 Archives of Microbiology (2022) 204:165 Page 3 of 10 165 to Li et al. (2014) and Khazi et al. (2018). The absorbance of growth and age of cultures, we fitted a model to the data Chlorophyll content was determined spectrophotometrically using interpolation in MATLAB software. at 665 nm according to Marker (1972). Physiological characterization Results To survey the photosynthetic activity and respiratory elec- Growth tron transport chains under different salinity and alkaline conditions, oxygen exchange was studied. Steady-state oxy- This study evaluated—in vivo experiments—the ability gen evolution was measured with a Clark-type electrode of acclimation and growth of Cylindrospermum sp. FS 64 PSII activity in whole cells. Cells cultured at temperature under multiple environmental factors at a short period of 30 ± 2 °C and constant illumination 60 μmol quanta m ­ −2 ­s−1 time under extreme DIC limitation. In general, the results (Inoue-Kashino et al. 2005). The amount of liberated oxygen supported the hypothesis that the combined environmental was normalized by the amount of chlorophyll according to factors can be led to growth and acclimation in different Poza-Carrion (Poza-Carrión et al. 2001). The initial physio- environmental conditions at short time. Comparison of the logical status of Cylindrospermum sp. FS 64 was performed growth curve of Cylindrospermum sp. FS 64 showed that by measuring the maximum photosynthetic rate (Pmax) and extreme alkaline condition (pH 11) was more favorable to photosynthetic efficiency (α) and light saturation (Ik) val- growth and had a significant effect (p < 0.05) on biomass ues after growth analysis. Photosynthesis–irradiance (P–I) production compared to pH 9 under extreme DIC limita- curves were calculated by measuring oxygen evaluation rates tion (Fig. 1). A study of the length of the incubation period during successive 1-min illumination periods with a step- revealed that 80 mM salinity caused significantly growth wise increase from 0 to 2500 μmol quanta ­m−2 ­s−1. The pho- increased at pH 11 after 48 h and, likewise no significant tosynthetic pigments were estimated in terms of chlorophyll a, phycocyanin, from 380 to 760 nm using Synergy HTX (Multi-Mode Microplate Reader, USA) and they normalized 0.30 17mM pH 9 to optical density according to Tang and Vincent (1999). 80mM 160mM The operation of photosystems and phycobilisomes charac- 0.25 teristics were analyzed spectrofluorimetrically according to 0.20 Inou-Kashino et al. (2005), Vermaas et al. (2008) and Zorz OD 750 et al. (2015). Room temperature fluorescence emission spec- 0.15 tra of the cells were recorded following Tiwari and Mohanty (1996) and Fraser et al. (2013). The excitation spectra were 0.10 recorded at λex: 440 to excite chlorophyll a and 550 nm for phycocyanin. The single-cell study (the fluorescence inten- 0.05 sity of single cell) which illustrated the physiological state of 24 48 72 96 the individual, live-cell and spectral unmixing (Grigoryeva Time (hours) and Chistyakova 2019) was measured using λ scan of confo- 0.35 cal laser microscope system (Leica TCS-SP5 CLSM—Leica pH 11 Microsystems Heidelberg GmbH, Mannheim, Germany). 0.3 Photosynthetic pigment excitation was carried out with an 0.25 argon laser at 405 nm. The fluorescence emission spectrum was collected by detecting wavelengths between 415 and 760 OD 750 0.2 (Ramírez et al. 2011; Sugiura and Itoh 2012; Shokravi and Bahavar 2021a, b). Analysis of the lambda scan data was 0.15 carried out using the Leica Confocal Software. 0.1 Statistical analysis 0.05 24 48 72 96 Time (hours) Analysis of variance (ANOVA) with the SPSS-24 software was used to evaluate the results. The ANOVA showed a sig- nificant difference between treatments with p < 0.05. All the Fig. 1  Variation of optical density (OD 750 nm) of Cylindrosper- mum sp. FS 64 under different salinity (17, 80 and 160 mM NaCl) experiments were carried out in six independent biologi- and alkaline pHs (9, 11) from 24 to 96 h. Line with error bars shows cal replicates. For relationships of photosynthesis activity, significant difference at P < 0.05 13 165 Page 4 of 10 Archives of Microbiology (2022) 204:165 effect was observed between 17 and 80 mM salinity at pH to examine the relationships and quantitative description 11 after 96 h. Regardless of salinity, pH 11 was the opti- between photosynthesis, growth, and age of culture (Fig. 3). mum pH in this strain up to 168 h (Result not shown). The We observed a significant increase in growth and biomass presence of 160 mM salinity at both alkaline pHs caused production and photosynthesis activity under the combined significant inhibition of growth and biomass production until environmental factors. A positive and significant correlation 96 h. The metabolic activities and synthesizing enzymes in was found under the combined 80 mM salinity and pH 11 the growth stage led to the shorter lag phase under alkaline after 96 h. pHs. Therefore, cells acclimatized in both combined condi- The combined effects of environmental factors on photo- tions 17 mM—pH 9, and 80 mM—pH 11 after 24 h. synthetic parameters (P–I) are summarized in Table 1. The maximum value of the photosynthesis activity (Pmax)— Photosynthesis (photosynthetic oxygen evolution indicating carboxylation or a step closely associated with and P–I curve) carboxylation—was approximately 90% higher at combined 80 mM salinity and pH 11 compared to pH 9. Ik indicat- Regardless of salinity and age of the cultures, the maxi- ing the irradiance at which control of photosynthesis passes mum rates of oxygen evolution of Cylindrospermum sp. FS from light absorption and photochemical energy conversion 64 gradually increased at pH 11 (~ 100–250 µmol O ­ 2 mg to reductant utilization (Sakshaug et al. 1997). Ik was lower −1 −1 Chl a × ­h ) against pH 9 (~ 120–160 µmol O ­ 2 mg Chl a−1 × ­h−1) (Fig. 2). The combined effect of salinity, alka- linity and age of culture revealed that the maximum rates of oxygen evolution significantly increased at pH 11 and 80 mM salinity after 48 h (Fig. 2b). In contrast, the signifi- cant decrease was observed under high salinity (160 mM) after 72 h at pH 9. Our purpose of the fitting model was 180 pH 9 17mM 80mM 160mM 160 Photosynthesis (oxygen evoluon) 140 (µmol O2 mg chl a -1×h-1) 120 100 80 60 40 Fig. 3  The fitting model of Cylindrospermum sp. FS 64 presented the 20 relation between the growth, photosynthesis and time under different 0 levels of salinity and alkalinity treatment. Each data-point represents 24h 48h 76h 96h different salinity and pH Time (hours) 300 Table 1  Comparison of parameters of photosynthesis–irradi- pH 11 ance curves (Pmax, the maximum photosynthetic rate (μmol ­O2 mg Photosynthesis (oxygen evoluon) 250 ­chl−1 ­h−1); α, photosynthetic efficiency (μmol ­ chl−1 ­h−1)/ O2 mg ­ (µmol O2 mg chl a -1×h-1) −2 −1 (μmol photon m ­ ­s ); Ik, light saturation point (µmquanta ­m−2 ­s−1) 200 of Cylindrospermum sp. FS 64 at different salinity and alkaline pHs after 72 h of inoculation 150 NaCl (mM) 17 80 160 100 Pmax—pH 9 67.41 ± 5.55 59.34 ± 4.22 52.45 ± 6.08 50 Pmax—pH 11 54.41 ± 3.05 74.34 ± 6.66 34.45 ± 3.18 0 α—pH 9 0.84 ± 0.04 0.85 ± 0.06 0.61 ± 0.16 24 48 76 96 α—pH 11 0.74 ± 0.02 0.87 ± 0.04 0.65 ± 0.1 Time (hours) Ik—pH 9 390 280 520 Ik—pH 11 260 120 440 Fig. 2  Comparison of photosynthetic oxygen evolution of Cylindros- permum sp. FS 64 at different salinity, pHs and time. Bars with error Values are means of three independent biological replicates ± stand- bars show significant difference at P < 0.05 ard deviation 13 Archives of Microbiology (2022) 204:165 Page 5 of 10 165 at the combination of low salinity (17 and 80 mM) and pH While no significant differences were observed between 17 11 compared to pH 9, indicating that the rate of water oxi- and 80 mM salinity at pH 9. In presence of 160 mM salin- dation in PSII was reduced. α is often used for comparing ity, the structure and stability of phycocyanin and PSII were the cyanobacteria shade endurance (shade tendency) under demolished at pH 11 after 72 h, although they are main- shading conditions. Noticeably, in the presence of 80 mM tained their structure up under pH 9. In addition, the absence salinity, the shade-adapted capacity of cyanobacterium sig- of the shift and stability of the Chl a of PSII was noticeable nificantly increased at both alkaline pHs. in all treatments. Absorption spectra PBS and PSII stability under different salinity and alkaline pHs In vivo absorbance spectroscopy as a common method to obtain an overview of the content and distribution of the pig- We investigated the distribution of energy between phyco- ments of cells (Fig. 4) showed that chlorophyll Soret bands biliproteins and PSII spectrofluorimetrically at excitation (~ 445 nm), chlorophyll a of PSII (~ 680 nm), and phycocya- 440 nm (chlorophyll- associated with PSII), and 550 (phy- nin (~ 620–630 nm) was the principal active compound in all cocyanin). We observed addition of 80 mM salinity was treatments. A shoulder at ~ 495 nm related to carotenoids has accompanied by increasing PSII (Fig. 5) and phycocyanin appeared. In addition, we observed an increase in absorption activity (Fig. 6) at pH 11 compared to pH 9. Although, of ~ 250–300 nm which is most probably related to one of increasing salinity (160 mM) drastically decreased the effi- the carotenoids bands (generally, organic molecules) under ciency and effectiveness of PSII and PC activity at both alka- the combination of pH 11 and 80 mM salinity. Our results line pHs after 24 h (Figs. 5, 6). This study confirmed the revealed that the dynamism and stability of PSII and phy- high growth, biomass production, and content of the PSII cobilisomes significantly increase in the combined effect of and PBS (absorption spectra) under the combined effect of 80 mM salinity and pH 11 compared to pH 9 after 72 h. 80 mM salinity at pH 11. 0.50 5000 17mM pH 9 17mM pH 9 0.45 80mM 4500 80mM 265 160mM 160mM 4000 0.40 Fluorescence intensity 3500 0.35 3000 Absorbance 0.30 2500 445 0.25 2000 490 0.20 630 685 1500 0.15 1000 0.10 500 0 0.05 640 660 680 700 720 230 280 330 380 430 480 530 580 630 680 730 Wavelength(nm) wavelength 0.55 10000 265 pH 11 pH 11 0.50 9000 0.45 8000 440 Fluorescence intensity 0.40 7000 490 685 628 Absorbance 0.35 6000 0.30 5000 0.25 4000 0.20 3000 0.15 2000 0.10 1000 0.05 0 230 280 330 380 430 480 530 580 630 680 730 640 660 680 700 720 wavelength Wavelength(nm) Fig. 4  Room temperature absorption spectra of in vivo Cylindrosper- Fig. 5  Comparison of the fluorescence intensity of Cylindrospermum mum sp. FS 64 cells adapted to different salinity and pHs for 72 h. sp. FS 64 at different salinity and pHs after 72 h of inoculation. Exci- Normalized to optical density (OD 750) tation 440 nm 13 165 Page 6 of 10 Archives of Microbiology (2022) 204:165 1200 120 pH 9 17mM pH 9 17 mM 80mM 680 80 mM 1000 160mM 100 160 mM Fluorescence intensity Fluorescence intensity 800 80 663 600 60 400 40 719 200 20 0 0 620 630 640 650 660 670 680 690 700 710 620 640 660 680 700 720 740 Wavelength(nm) Wavelength (nm) 180 1600 pH 11 pH 11 679 160 663 1400 140 Fluorescence intensity Fluorescence intensity 1200 120 1000 649 100 800 80 600 714 60 400 40 200 20 0 0 620 630 640 650 660 670 680 690 700 710 620 640 660 680 700 720 740 Wavelength(nm) Wavelength (nm) Fig. 6  Comparison of the fluorescence intensity of Cylindrospermum Fig. 7  Fluorescence spectra of individual cell (Lambda scan) of sp. FS 64 at different salinity and pHs after 72 h of inoculation. Exci- Cylindrospermum sp. FS 64 at different salinity and pHs after 72 h of tation 550 nm inoculation. λexc = 405 nm Spectroscopic study of a single cell by CLSM single-cell study confirmed the highest growth and stability of PS and PBS depends on combined 80 mM salinity and Investigation of single cell illustrated the physiological state pH 11 (Figs. 1, 5, 6). of the individual, live-cell and maximum fluorescent of pig- ment protein which enabled us to monitor the dynamic pro- cesses of the chosen cells as spectral unmixing and all steps Discussion of the energy transfer chain (Grigoryeva and Chistyakova 2019; Shokravi and Bahavar 2021b) (Fig. 7). We observed Overall, our results provide important insight that combined the high fluorescence of Chl a (PSII) at ~ 680 nm and PSI multiple factors such as salinity, alkalinity, and the age of the at ~ 715 nm at vegetative cells compared to heterocyst— cultures in laboratory conditions plays a key role in accli- because of low amounts of PSII and PBS at heterocyst matizing the growth, and photosynthesis of Cylindrosper- (heterocyte result not shown). In addition, a clear shoulder mum sp. FS 64. Absorption spectra and chlorophyll con- was observed around 663 nm related to the APC (Allophy- centration (OD 750) methods as an overview of growth and cocyanin) of phycobilin production, which was highest at cells activity on the culture (Schulze et al. 2011) indicated 80 mM salinity at pH 11. Increasing salinity (160 mM) led that salinity and alkalinity (combine together—pH 11 and to declined APC content at both alkaline pHs. This reduc- 80 mM salinity) cannot be considered as stress to limit the tion which was saline dependent caused the demolished growth (Borowitzka 2018) of Cylindrospermum sp. FS 64. structure of PBS at pH 9 compared to pH 11. Chl a (PSII- Low salinity (17 and 80 mM) in heterocystous cyanobacte- 680 nm) was the most stable pigment under all conditions, ria (Srivastava et al. 2009) can be desired as nutrition and and it was higher under the combined 80 mM salinity and stimulant leads to a significant increase in growth, biomass pH 11. No significant difference of PSII and also PSI activity production (Miriam et al. 2017), and photosynthesis opera- was observed between 17 and 80 mM salinity after 72 h at tion (Singh et al. 2015). The elevated salinity (160 mM) at pH 9 against pH 11. While the highest PSI activity belongs both alkaline pHs 9 and 11 led to inhibition of Chl biosyn- to 80 mM salinity at pH 11. Overall, our results of the thesis (Chris et al. 2006) which resulted in a decrease in 13 Archives of Microbiology (2022) 204:165 Page 7 of 10 165 chlorophyll pigment. Besides, these findings are in line with Cylindrospermum sp. FS 64 can be ascribed to the different our other study on Nostoc sp. UAB 206 that isolated from media, pH, and salinity used, implying an increase in energy the Spanish paddy field (not published data). Valiente and for growth. Leganes (1990), Poza-Carrión et al. (2001), Soltani et al. To better understanding PBS and PS activity and stabil- (2006), Amirlatifi et al. (2018), Abbasi et al. (2019) and ity, we have used the fluorescence assay. The strain showed Shokravi and Bahavar (2021a) have indicated that the opti- nearly 90% of PBS and PSII stability under the combination mum pH for growth, photosynthesis and nitrogen fixation of 80 mM salinity and pH 11 after 96 h compared to pH of terrestrial cyanobacteria under combined environmental 9. While increasing salinity (160 mM) led to demolished factors is about 8 or 9. The response of strain to pH 11 and of the PBS and PSII structure at both alkaline pHs after 80 mM salinity may depend on the high genetic plasticity 24 h. Galetović et al. (2020) reported most research focuses (Boussiba et al. 2000) or and is an inherent characteristic on PBS behavior and stability in different temperature and (Tang and Vincent 1999) which resulted in without any pH 5–7 (Antelo et al. 2008), pH 2.0, 6.5 and 8.0 (Couteau requirement for an acclimation process (Vonshak and Tor- et al. 2004) and pH range of 4–9 (Leu et al. 2013). Chris zillo 2007). Furthermore, we observed a strong correlation- et al. (2006) found a decrease in growth, chlorophyll content, fitting model—under the combined 80 mM salinity and carotenoid, phycocyanin, and PS II activity of Cylindros- pH 11 up to 96 h which confirmed the highest activity of permum sp. due to individual salinity as well as in combi- cells under this condition. Pigment analysis of cell cultures nation with UV-B treatments. In addition, Srivastava et al. (absorption spectra) also confirmed the results of the activity (2009) investigated that 150 mM salinity and pH 7.5 caused of the cells under this condition. a decline in PSI, PS II, and whole chain activities in Ana- No accurate understanding of the mechanisms involved baena doliolum after 24 h. The difference between these in pH homeostasis in cyanobacteria. Giraldez-Ruiz et al. findings may be due to the use of various pH ranges which (1997) proposed that Na, K, and Ca could be considered as influences the growth, metabolism, regulation, and distribu- part of the pH homeostatic system. Touloupakis et al. (2016) tion of cyanobacteria (Jin and Kirk 2018). reported that many mechanisms have been suggested for pH During cultivation, chlorophyll content decreases due to homeostasis and the regulation of ­CO2/HCO3 concentration. environmental factors or dying of the part of the population Most carbon sources are in bicarbonate ions under severe (age of culture). (Schulze et al. 2011; Shokravi and Bahavar carbon dioxide deficiency in alkaliphilic cyanobacteria 2021a, b). Therefore, single-cell study is a new method that (Boyd 2015). The carbon dioxide concentration mechanism provide information on the dynamic behavior of each cell (CCM) is the key process that enables them to acclimate to (Sugiura and Itoh 2012; Grigoryeva and Chistyakova 2019). alkaline conditions (Klanchui et al. 2017). The operation By confocal laser microscopy, Ying et al. (2002), Wolf and of CCM involves a high amount of energy and naturally Schüßler (2005) and Sugiura and Itoh (2012) demonstrated requires a high operation of photosynthesis—along with different fluorescence spectra of vegetative and heterocyte other needs—and high efficiency of PSI, PSII, and PBS cells-unmixing spectra. The results of single-cell spectra (Mangan and Brenner 2014). Regarding the highest growth support that combination 80 mM salinity and pH 11 up to and photosynthesis at pH 11, Cylindrospermum sp. FS 64 96 h led to a noticeable increase and stability in all parts of have a powerful carbon dioxide concentration mechanism the phycobilisome and PSII activity. This stability of PSII and flexibility to induce. may depend on the physical change in enzymes and binding The main reasons of growth measuring is understanding sites in PSII and potent PSII efficiency. Reduction of PBS the balance between photosynthesis and respiration (Nygård and PSII content indicating the degradation of their structure and Dring 2008). The oxygen liberation (a marker of PSII by increasing salinity at both alkaline pHs. We observed a activity) analysis confirmed the growth results. Regardless decline and the shifted peak of PSI (719 nm) that is affected of salinity, the maximum photosynthesis (Pmax) of Cylin- by the lower PC content at pH 9 compared to pH 11. There- drospermum sp. FS 64 was higher at pH 9 against pH 11. fore, cells might accept less excitation energy when PC is Addition salinity (80 mM) caused an increase in Pmax (Ye reduced (Schmitt et al. 2020). and Gao 2004; Dhiab et al. 2007) at pH 11 which can be attributed to the high efficiency of water oxidation in PSII. In contrast, 80 mM salinity led to decreasing in saturating Conclusion irradiance and increased shade-adapted capacity of strain at both alkaline pHs, which influenced an increase in the In conclusion, the different methods confirmed that combin- relative content of PSII activity and the antenna size of PS ing environmental factors (different alkaline pH, salinity, II (Inoue-Kashino et al. 2005). Briand et al. (2004) reported and time under extreme DIC limitation) can affect cyano- that Ik is the most reliable parameter for assessing and com- bacterial behaviors individually or in combination and led paring the variable-light requirement. The high Ik value of to regulation and acclimation in short time. We observed 13 165 Page 8 of 10 Archives of Microbiology (2022) 204:165 Cylinrospermum sp. FS 64 acclimatized through different Bemal S, Anil AC (2018) Effects of salinity on cellular growth and strategies and has developed a mechanism for the highest exopolysaccharide production of freshwater Synechococcus strain CCAP1405. J Plankton Res. https://d​ oi.o​ rg/1​ 0.1​ 093/p​ lankt/f​ bx064 growth, photosystems operation, phycobilisomes activity Borowitzka MA (2018) The ‘stress’ concept in microalgal biol- and light-saturated photosynthetic under 80 mM salinity ogy—homeostasis, acclimation and adaptation. J Appl Phycol and pH 11. Conversely, elevated salinity was time depend- 30(5):2815–2825. https://​doi.​org/​10.​1007/​S10811-​018-​1399-0 ent at both alkaline conditions. Several lines of evidence Bouazzara H, Benaceur F, Chaibi R, Boussebci I, Bruno L (2020) Combined effect of temperature, pH and salinity variation on the supported this issue. From an applied point of view, this growth rate of Gloeocapsa sp. in batch culture method using Aiba cyanobacterium can be used in alkaline–saline paddy fields and Ogawa medium. Eurasian J Biosci 14(2):7101–7109 and agricultural lands as a biofertilizer, soil conditioners, Boussiba S, Wu X, Zarka A (2000) Alkaliphilic cyanobacteria. In: and other biotechnological purposes. Seckbach J (ed) Journey to diverse microbial worlds. Cellular Origin and Life in Extreme Habitats, vol 2. Springer, Dordrecht. https://​doi.​org/​10.​1007/​978-​94-​011-​4269-4_​15 Acknowledgements The authors would like to appreciate the kind Boyd CE (2015) pH, carbon dioxide, and alkalinity. In: Water qual- collaboration of Professor Eduardo Fernandez Valiente (Universidad ity. Springer, Cham, pp 153–178. https:// ​ d oi. ​ o rg/ ​ 1 0. ​ 1 007/​ Autónoma de Madrid -UAM) and Professor Neda Soltani (Shahid 978-3-​319-​17446-4_8 Beheshti University, Iran). This work was supported in part by Doctor Briand JF, Leboulanger C, Humbert JF, Bernard C, Dufour P (2004) Enrique Llobet Lleó to assist the world microbial community. Cylindrospermopsis raciborskii (Cyanobacteria) invasion at mid- latitudes: selection, wide physiological tolerance, or global warm- Funding Open Access Funding provided by Universitat Autonoma de ing? J Phycol 40(2):231–238. https://d​ oi.o​ rg/1​ 0.1​ 111/j.1​ 529-8​ 817.​ Barcelona. 2004.​03118.x Chittora D, Meena M, Barupal T, Swapnil P (2020) Cyanobacteria as a Declarations source of biofertilizers for sustainable agriculture. Biochem Bio- phys Rep 22:100737. https://d​ oi.o​ rg/1​ 0.1​ 016/j.b​ brep.2​ 020.1​ 00737 Chris A (2012) Effect of nickel stress on growth and antioxidants in Conflict of interest I and Shadman Shokravi have no conflict of inter- cyanobacterium Cylindrospermum sp. Asian J Bio Sci 7(1):13–17 est to declare. 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