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

Vol.:(0123456789) 1 3 h ps://doi.o g/10.1007/s10811-022-02693-3 Glycogen, poly(3‑hyd oxybu y a e) andpigmen accumula ion in h ee Synechocys is s ains whenexposed oas epwise inc easing sal s ess K.Meixne 1,2· C.Da e 1· D.Dalnoda 1· K.M ázo á3· K.H ubano á3· V.K zyzanek3· J.Nebesa o a4,5· O.Samek3· Z.Šed lo á6· E.Slanino a6· P.Sedláček6· S.Ob uča6· I.F i z1 Recei ed: 21 Sep embe 2021 / Re ised and accep ed: 7 Janua y 2022 © The Au ho (s) 2022 Abs ac The cyanobac e ial genus Synechocys is is o pa icula in e es o science and indus y because o i s e icien pho o ophic me abolism, i s accumula ion o he polyme poly(3-hyd oxybu y a e) (PHB) and i s abili y o wi hs and o adap o ad e se g owing condi ions. One such condi ion is he inc eased salini y ha can be caused by ecycled o b ackish wa e used in cul i a ion. While o e all educed g ow h is expec ed in esponse o sal s ess, o he me abolic esponses ele an o he e iciency o pho o ophic p oduc ion o biomass o PHB (o bo h) ha e been expe imen ally obse ed in h ee Synechocys- is s ains a s epwise inc easing sal concen a ions. In esponse o ecen epo s on me abolic s a egies o inc ease s ess ole ance o he e o ophic and pho o ophic bac e ia, we ocused pa icula ly on he s ess-induced esponse o Synechocys is s ains in e ms o PHB, glycogen and pho oac i e pigmen dynamics. O he h ee s ains s udied, he s ain Synechocys is c . salina CCALA192 p o ed o be he mos ole an o sal s ess. In addi ion, his s ain showed he highes PHB accumula ion. All he h ee s ains accumula ed mo e PHB wi h inc easing salini y, o he poin whe e hei pho osys ems we e s ongly inhibi ed and hey could no longe p oduce enough ene gy o syn hesize mo e PHB. Keywo ds Sal s ess· Synechocys is sp.· Poly(3-hyd oxybu y a e)· Glycogen· Pigmen s In oduc ion Cyanobac e ia ha e a long e olu iona y his o y in which hey colonized a wide a ie y o habi a s, anging om glacie s o ho sp ings, om eshwa e o saline en i on- men s. They e ol ed di e en s a egies o cope wi h en i- onmen al s ess and hey manage o adap o changing en i- onmen al condi ions wi hin hei habi a s. Cyanobac e ia a e capable o acclima ing o a ious s ess condi ions, by adap ing hei physiological, biochemical and molecula ac i i ies. They espond o empe a u e s ess by al e ing memb ane composi ion o adap luidi y h ough sa u a- ion o a y acids and p o ein in eg i y. S a egies used in esponse o UV s ess include sca enging eac i e oxygen species, syn hesis o UV-abso bing/shielding compounds, Highligh s • Synechocys is sp. adap ed o an o he wise le hal salini y o 6.5 %. •Pigmen amoun s and ela ions changed wi h inc easing salini y. •In s essed cells inc eased PHB bu dec eased glycogen amoun s occu ed. •The highes amoun o PHB accumula ed in Synechocys is salina CCALA192 a 4.5 % NaCl. * K. Meixne Ka ha [email p o ec ed] 1 Ins i u e o En i onmen al Bio echnology, Depa men o Ag obio echnology, IFA-Tulln, Uni e si y o Na u al Resou ces andLi e Sciences, Vienna, Kon ad-Lo enz-S aße 20, 3430Tulln, Aus ia 2 BEST Bioene gy andSus ainable Technologies GmbH, In eldgasse 21b, 8010G az, Aus ia 3 Ins i u e o Scien i ic Ins umen s, The Czech Academy o Sciences, K álo opolská 147, 61264B no, CzechRepublic 4 Ins i u e o Pa asi ology, Biology Cen e, The Czech Academy o Sciences, B aniso ska 31, 37005CeskeBudejo ice, CzechRepublic 5 Facul y o Science, Cha les Uni e si y, Vinicna 7, 12844P ague2, CzechRepublic 6 Facul y o Chemis y, B no Uni e si y o Technology, Pu kyno a 118, 61200B no, CzechRepublic / Published online: 30 Ma ch 2022 Jou nal o Applied Phycology (2022) 34:1227–1241 1 3 epai o damaged DNA and esyn hesis o p o eins. Agains me al-induced s ess, cyanobac e ia p o ec hemsel es by al e ing hei memb ane s uc u e and ac i a ing o inac- i a ing ans e pumps. Besides ha , sal s ess is a majo abio ic s ess p oblem in a id and semi-a id egions as well as in i iga ion a eas (Sudhi and Mu hy 2004). Salini y educes he a ailabili y o wa e and inc eases he Na+ and Cl− concen a ion. Cyanobac e ia educe he Na+-up ake as well as hei ac i e e lux ia Na+/H+ an ipo e , hey induce o ganic compounds, inc ease an ioxida i e de ense sys ems o de oxi y he eac i e oxygen species, accumula e com- pa ible solu es o compensa e osmo ic p essu e and inally, also exp ess sal -inducible p o eins o cope wi h sal s ess (Klähn and Hagemann 2011; Rezayian e al. 2019). Besides hese su i al s a egies, cyanobac e ia, such as Synechocys is sp., o he e o ophic bac e ia, such as Cup ia idus neca o accumula e ca bon and ene gy s o age polyme s. I is known ha he lipophilic poly(3-hyd oxy- bu y a e) (polyhyd oxybu y a e,PHB) is o med unde ni ogen and/o phospho us de iciency wi h simul ane- ous excess o ca bon and ene gy. PHB is known o ac in he e o ophic bac e ia as a p o ec an agains osmo ic and oxida i e s ess, agains pH luc ua ions and inc eased p es- su e (Ob uca e al. 2020, 2021; F i z e al. 2021). Besides PHB, cyanobac e ia p oduce he hyd ophilic polyme gly- cogen, which is inc easingly syn hesized du ing he s a- iona y phase (Velmu ugan and Incha oensakdi 2018) and is s a ed o be a he a quick- esponse ene gy and ca bon s o age o cope wi h da kness (nigh ), when i s deg ada- ion may be he only a ailable ene gy sou ce o he s ic pho o ophic cells (Dam ow e al. 2016; Koch and Fo ch- hamme 2021). Fu he mo e, glycogen accumula ion is c ucial o phycobilisome deg ada ion and assembly o he hylakoid memb ane in p esence o ligh (Velmu u- gan and Incha oensakdi 2018). I is no ully unde s ood why cyanobac e ia p oduce PHB and glycogen in pa allel, as bo h s o age compounds compe e o he ca bon pool (Wu e al. 2002; Rueda e al. 2020; F i z e al. 2021). The glycogen syn hesis pa h om CO2 s a s wi h 3-phospho- glyce a e, which can also be con e ed in o py u a e, hen in o ace yl-CoA and inally in o PHB (Rueda e al. 2020). Howe e , PHB and glycogen le els a e independen o each o he (Velmu ugan and Incha oensakdi 2018; Koch e al. 2019). Glycogen concen a ions in Synechocys is PCC6803 a e s a ed o ange om 18.5% (Velmu ugan and Incha - oensakdi 2018) up o 50%(pe cell d y weigh , CDW) (Luan e al. 2019). The concen a ion ises wi h cul i a- ion and peaks a he mid-s a iona y phase (22.7%CDW), addi ionally i inc eases unde ni ogen de iciency (36.8% CDW) and glucose addi ion (41.3% CDW) (Monshupanee and Incha oensakdi 2014). So does PHB, whose maxima a e epo ed o be abou 10–20% (CDW) in chlo o ic cells and can be u he inc eased by gene ic enginee ing o 63% when cul i a ed in ni ogen- and phospho ous-deple ed medium and o 81% when ace a e is added (Koch e al. 2020). Ini ial concen a ions o glycogen and PHB in Syn- echocys is PCC6803 a e epo ed o be app oxima ely 12% and 1%, espec i ely (Du and S i as a a 2018). The expe imen s conduc ed he ein aimed a e alua ing o wha ex en Synechocys is sp. can su i e and adap o s epwise inc easing sal concen a ions and how biomass concen a ion changes, especially in ega d o glycogen, PHB and pigmen s. Fo hese pu poses, g ow h and cell composi ion o h ee Synechocys is s ains we e moni o ed a s epwise inc easing sal (NaCl) concen a ions (0% o 8%). Ma e ial andMe hods S ains andMedia The s ains Synechocys is sp. PCC6803, Synechocys is c . salina CCALA192 and an isola ed wild- ype Synechocys- is s ain, called IFA3, we e used. PCC6803 was ob ained om he Pas eu cul u e collec ion (FRA) and CCALA192 om he cul u e collec ion o au o ophic o ganisms (CZE). IFA3 was isola ed in 2018 om a pond in Lowe Aus ia and iden i ied by sequencing i s 16S- DNA (see supplemen a y ma e ial). The s ains we e cul i a ed in a mine al medium based on BG-11 (Rippka e al. 1979), whose ni ogen and phos- pho ous con en s we e adjus ed o allow an ea ly biomass g ow h un il s a a ion o ni ogen and phospho ous ini ial- izes PHB p oduc ion wi hin a single cul i a ion s age. Ni o- gen limi a ion is indica ed by a ypical colo swi ch om blue-g een o oli e-g een and o ange. Composi ion mine al medium pe li e : 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, ace elemen solu ion 1.50mL. Composi ion ace elemen solu ion pe li e 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, KC (SO4)2.12H2O: 0.470g. Expe imen al se up In he he ein conduc ed expe imen s, he sal (NaCl) concen- a ion in he medium was s epwise inc eased. Cul i a ion o each s age was ca ied ou in 200mL mine al medium in 500-mL E lenmeye lasks in o de o achie e a hin laye hickness o he cul u es. The lasks we e shaken manually wo imes pe day. Fo cul i a ion ambien CO2 concen a- ion (419ppm – (McGee 2021)), 22°C, 75.9 ± 9.5µmol 1228 Jou nal o Applied Phycology (2022) 34:1227–1241 1 3 pho ons m−2 s−1 (me al halide lamp, Philips Mas e HPI-T Plus, 250W) and a day/nigh cycle o 16/8h we e p o ided. The expe imen s we e ca ied ou as iplica es, each cul i- a ion s age las ed 42days. Cells we e ans e ed om he cu en o he nex s age be ween weeks wo and ou (see a ows in Fig.1), depending on cell densi y (OD750). The OD750 a which he inoculum was aken was 2.20 ± 0.37 o PCC6803, 2.36 ± 0.39 o CCALA192 and 1.98 ± 0.74 o IFA3. Fo he inocula ion, abou 40mL o cul u e om each lask o he cu en s age we e ha es ed (pooled om ip- lica es) and cell numbe s we e coun ed ia Thoma chambe . All o he suspension was cen i uged gen ly a 800×g, o 15min, he cells we e esuspended in he necessa y amoun o sal -con aining mine al medium o ge a cell densi y o 1.2–2 × 108 mL−1. The eo , 20mL we e ans e ed in o 180mL medium o he nex s ep, esul ing in 1.2–2 × 107 cells mL−1. The NaCl concen a ion o he s ages was se o 0% (w/ , e e ence), 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), espec i ely. G ow h moni o ing andBiomass analysis E apo a ion was compensa ed by adding he necessa y amoun o deionized wa e o each lask be o e samples we e aken. G ow h was moni o ed ia op ical densi y a 435nm (OD435) and 750nm (OD750) wice a week, also pH was measu ed in hose samples. Once pe week, he composi ion o he biomass was analyzed. The e o e, cells we e sepa a ed om he cul i a ion media and washed wi h e e se osmosis wa e by cen i uga ion. Aliquo s o we biomass we e s o ed a -20°C un il analysis o gly- cogen and pigmen s, and aliquo s we e d ied a 105°C o de e mina ion o cell d y weigh (CDW) and PHB. The cell numbe o each sample was es ima ed by spec al low cy ome e (Cy ek Au o a) whe e samples we e indi- idually washed and p ope ly dilu ed by phospha e bu e (50mM, pH 8). Fo analyzing he PHB con en , he d ied pelle was diges ed wi h 200 µL concen a ed (98%) sul u ic acid (H2SO4) o 30min a 90°C (adap ed a e (Ka e al. 1983)). In his s ep, PHB was con e ed o c o onic acid. A e wa ds, he samples we e illed o 10mL wi h deion- ized wa e and p epa ed o HPLC (high-pe o mance liquid ch oma og aphy) analysis (Agilen 1100; column: T ansgenomic COREGEL 87H3; de ec o : Agilen 1100 RI). The glycogen con en o he cells was de e mined based on Koch e al. (2019). The pelle o 2mL sample was esuspended in po assium hyd oxide (KOH, 30% w/ ) and incuba ed a 95°C o 2h. Ice cold e hanol (absolu e) was added and he sample was incuba ed a -20°C o e nigh o p ecipi a e glycogen. Subsequen ly, he pelle was washed wice wi h e hanol (1s wash: 70%, 2nd wash: absolu e), d ied a 60°C, esuspended in sodium ace a e bu e (100mM, pH 4.5), and diges ed wi h amyloglucosidase solu ion (0.62 U µL−1) a 55°C and con inuous shaking o 2h. Ca ez p ecipi a ion (me hod o emo ing u bidi y such as col- loids (Gänzle 2021)) was ca ied ou o cla i y he liquid and emo e he enzyme. Finally, he sample was p epa ed o HPLC analysis (same con igu a ion as o PHB analysis). Fo analyzing con en s o chlo ophylla and o al ca o e- noids (ca o enoids) as well as c-phycocyanin (phycocyanin), he washed we biomass was ex ac ed wi h e hanol (absolu e) (Lich en hale and Wellbu n 1983; Ri chie 2006) and e e se osmosis wa e , espec i ely. Abso bances o he ex ac s we e measu ed wi h a UV–Vis spec ome e (Shimadzu UV-1800). Pigmen concen a ions we e calcula ed om he abso bance alues o he ex ac s a ce ain wa eleng hs (chlo ophylla: 665 nm and 750 nm (Ri chie 2006); o al ca o enoids: 470 nm and 750 nm, modi ied a e Lich en hale and Wellbu n (1983); c-phycocyanin: 615 nm and 652 nm (Benne and Bogo ad 1973)) wi h equa ions (1-3): Fig. 1 Time cu e o he conduc ed expe imen s. Cells om he cu en s age we e ans e ed o he nex s age be ween weeks wo and ou (a ows) 1229Jou nal o Applied Phycology (2022) 34:1227–1241 1 3 Ex ac ion-based pigmen analysis was supplemen ed by hei spec oscopic de ec ion di ec ly in cyanobac e ial cell suspensions using di usi e ansmission spec opho ome y. Fo his pu pose, undilu ed samples we e measu ed in silica cu e es on UV–Vis spec opho ome e Hi achi U-3900H wi h in eg a ing sphe e 60mm DIA a scan speed 600nm min−1. Ul as uc u e Fo ul as uc u al analysis, cyanobac e ial cul u es we e cen i uged (4min, 1000×g). Samples o c yogenic scan- ning elec on mic oscopy (c yo-SEM) we e p epa ed by pipe ing he concen a ed pelle o cells on 6-mm Al ca - ie ype A and closing wi h he la side o ype B. Samples we e ixed using he high-p essu e eezing me hod (EM ICE, Leica Mic osys ems) wi hou using any c yo-p o ec - an . F ozen samples we e ans e ed in o a c yo- acuum p epa a ion chambe (ACE600, Leica Mic osys ems) and hen unde wen eeze ac u ing and sublima ion a -95°C o 7min. No me al coa ing was applied. Following subli- ma ion, cyanobac e ial cells we e obse ed in a scanning elec on mic oscope (Magellan 400/L, FEI) equipped wi h a c yo s age a -120°C using a 1–2keV elec on beam. Fo ansmission elec on mic oscopy (TEM), he con- cen a ed pelle o cells was pipe ed on 3-mm Al ca ie s co e ed wi h 1% solu ion o leci hin in chlo o o m and ixed using he high-p essu e eezing me hod (EM ICE, Leica Mic osys ems). F ozen samples we e hen ans e ed in o a eeze-subs i u ion uni (AFS2, Leica Mic osys ems). Sub- s i u ion solu ion con ained 1.5% OsO4 in ace one, he p o o- col used o he eeze-subs i u ion was p e iously desc ibed in Kouřilo á e al. (2021). A e eeze subs i u ion, samples we e washed h ee imes in ace one o 15min each and g adually in il a ed wi h medium-ha dness epoxy esin (Epoxy embedding medium, Sigma-Ald ich). In il a ion mix u es o epoxy esin and ace one in a ios 1:2, 1:1, 2:1, 1:0 we e changed a e 1h. A e he inal exchange, samples we e le in esh pu e esin unde a acuum in a desicca o o e nigh . Samples in esh esin we e cu ed using 62°C hea o 48h and cu o ul a hin sec ions on ul amic o ome (Ul acu UCT, Leica Mic osys ems) using a diamond kni e (Dia ome) wi h 45° cu ing angle. Sec ions on 300 mesh (1) Chlo ophylla[ mg L− 1] =11.90355 ∗ ( A665 −A750 ) (2) To al ca o enoids [mg L−1]= 1000 ∗ ( A470 −A750 ) −2.05 ∗chlo ophylla [ mg L− 1] 245 (3) C −phycocyanin[mg L−1]= A 615 − 0.474 ∗ A 652 5.34 ∗1000 coppe g ids we e hen s ained using solu ions o u anyl ace a e and lead ci a e and inally obse ed in ansmission elec on mic oscope JEOL 1010 using accele a ing ol age 80kV and images we e digi ally eco ded by CCD came a Mega iew III (Olympus). Resul s andDiscussion Th ee Synechocys is s ains (PCC6803, CCALA192, IFA3) we e exposed o s epwise inc easing sal concen a ions o e alua e how a hey a e able o adap o inc easing sal con- cen a ions ( om 0 o 8% w/ NaCl) and how biomass com- posi ion changes. The g ow h o CCALA192 (OD750) in all sal concen a ions is shown as an example in Fig.2 ( hose o PCC6803 and IFA3 a e in he supplemen a y ma e ial). Sal concen a ions o 0%, 3%, 6% and 8% (shown as solid lines) we e chosen o illus a e he e ec on g ow h and biomass composi ion (Figs.3, 4, and 5). These s ages ep esen no (0%, e e ence), mode a e and high sal concen a ions. The in e media e sal concen a ions we e necessa y o g adually adap he cells o he inc easing salini y. The esul s o hese s ages do no show ou s anding alues no do hey p o ide di e en insigh s. The e o e, we e ained om p esen ing all da a in he main sec ion o his manusc ip . The whole da a (g ow h cu es a OD750, OD485 [-], OD750[-], pH [-], CDW [g L−1], PHB [mg L−1], glycogen [mg L−1], chlo ophylla [mg L−1], phycocyanin [mg L−1], o al ca o enoids [mg L−1]) can be ound in he supplemen a y ma e ial. G ow h The cell d y weigh (CDW) o all he h ee Synechocys is s ains (PCC6803: Fig.3a, CCALA192: Fig.4a, IFA3: Fig.5a) was highes in he e e ence cul i a ions, wi hou sal addi ion (0% NaCl). CCALA192 demons a ed he highes biomass concen a ions (2.08 ± 0.04g L−1) ollowed by IFA3 (1.98 ± 0.07g L−1) and PCC6803 (1.85 ± 0.03g L−1). Fo all h ee s ains, i is clea ha g ow h dec eased wi h inc easing sal concen a ions and s agna ed a a sal concen a ion o 8%. In he medium wi h 3% sal he inal biomass concen- a ions o all h ee s ains we e compa able – 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 he medium wi h 6% sal IFA3 showed he highes biomass concen a ions (1.11 ± 0.05g L−1), ol- lowed by CCALA192 (1.06 ± 0.09g L−1) and PCC6803 (0.90 ± 0.01g L−1). In 8% NaCl only IFA3 showed sligh g ow h and eached 0.39 ± 0.03g L−1 a e a cul i a ion ime o 42days while bo h, CCALA192 and PCC6803, did no show any g ow h. T ends in cell numbe s o cul- u es o CCALA192 and PCC6803 (42days o cul i a ion), 1230 Jou nal o Applied Phycology (2022) 34:1227–1241 1 3 de e mined by low cy ome y (da a no shown) and CDW, espec i ely, co esponded o each o he . As expec ed, he exposi ion o cyanobac e ial cul u es o s epwise inc easing salini ies in cul i a ion media pa ially inhibi ed he g ow h o he cells, wi h he inhibi o y e ec being mo e p onounced he highe he sal concen a ion in he cul u e medium. Velmu ugan and Incha oensakdi (2018) s a ed ha g ow h o mode a ely halo ole an cyanobac e- ia Synechocys is is ha dly a ec ed up o 100mM (0.6%) NaCl and esul s in a biomass concen a ion o 1.4g L−1. Wi h inc easing sal concen a ions, biomass concen a ions declined om 1.3g L−1 (200mM = 1.2% NaCl) o 1.1g L−1 (300mM = 1.8% NaCl) (day 20). These alues (achie ed a 100µmol pho ons m−2 s−1, 28°C, in BG-11 medium) a e wi hin he ange o hose achie ed by ou expe imen s (0.61 o 0.96g L−1 a 2%NaCl and day 21, 1.55 o 2.04g L−1 a 2% NaCl and day 42). Also, achie ed esul s abou biomass we e con i med by da a om low cy ome e , i.e., cul u es wi h inc easing sal concen a ions demons a ed dec easing end also in cell numbe (da a no shown). PHB All h ee s ains p oduced PHB. The cellula concen a- ion o PHB inc eased o e cul i a ion ime (PCC6803: Fig.3b, CCALA192: Fig.4b, IFA3: Fig.5b). In he e - e ence cul i a ions (0% NaCl), lowes PHB concen a ions we e achie ed. The PHB con en inc eased up o a ce ain sal concen a ion, which was indi idually di e en o each s ain and was hen dec easing when salini y u he ose. The highes PHB concen a ion (6.98 ± 0.10% CDW) was achie ed by CCALA192 in 4% sal a e 42days o cul i- a ion, ollowed by PCC6803 (3.60 ± 0.28% CDW) in 3% and IFA3 (3.27 ± 0.8% CDW) in 2%, a e 42 and 36days, espec i ely. The same end is isible when compa ing PHB concen a ions o he s ains a e he same cul i a ion ime (42days) in 3% sal —CCALA192: 4.43 ± 0.10% CDW, PCC8603: 3.60 ± 0.28% CDW, IFA3: 2.33 ± 0.19%CDW. A all he sal concen a ions, CCALA192 accumula ed highe PHB amoun s han he o he wo s ains. Fig. 2 G ow h cu es o all expe imen s conduc ed wi h Synechocys is CCALA192. The solid lines show sal concen a ions o 0%, 3%, 6% and 8% and a e desc ibed in mo e de ail in he ex . The a ows indica e he inocula ion o he nex s age. n = 3 1231Jou nal o Applied Phycology (2022) 34:1227–1241 1 3 The PHB con en s o all he h ee s ains in he e e ence cul i a ions (0% NaCl) we e by a lowe (0.21 o 0.61% CDW, day 21) han epo ed o Synechocys is PCC6803 (abou 2.5% CDW, day 20, a 100µmol pho ons m−2 s−1, 28°C, in BG-11 medium) (Velmu ugan and Incha oen- sakdi 2018) and o ni ogen deple ed cells (abou 15–16.5% CDW, day 12, a 150µmol pho ons m−2 s−1, 30°C, in BG-11 medium + 0.4%( / ) ace a e) (Du and S i as a a 2018). PHB concen a ion inc eased wi h inc easing cul i a ion ime bu did no exceed 2% CDWin PCC6803, when cul i- a ion was s opped on day 42. Reasons could be he lowe empe a u e and ligh in ensi y in ou expe imen s lead o lowe g ow h compa ed o li e a u e. And mos likely ha ni ogen dep i a ion occu ed la e han day 20 o 12, due o he used medium, which we adap ed o single-s age cul i a- ion. Fu he mo e, no o ganic ca bon was added as desc ibed by Du and S i as a a (2018). IFA3 and CCALA192 con- ained 2.1% and 2.7% CDW a he end o cul i a ion. Glycogen Glycogen concen a ions (PCC6803: Fig.3b, CCALA192: Fig.4b, IFA3: Fig.5b) we e—compa able o PHB concen a ions—inc easing in all he h ee s ains wi h inc easing cul i a ion ime. Ano he simila i y o PHB accu- mula ion was ha CCALA192 also had he highes glyco- gen concen a ions bu in con as in 3% NaCl. The second highes glycogen concen a ion was achie ed by PCC6803, ollowed by IFA3. In all he s ains he highes glyco- gen le els we e accumula ed in 3% NaCl—CCALA192 (49.43 ± 0.58% CDW) a e 42, by PCC6803 (48.60 ± 1.82% CDW) and IFA3 (47.24 ± 0.73% CDW) a e 35days. Wi h u he inc easing sal concen a ions glycogen le - els dec eased. This e ec is clea ly isible la e in cul i a- ion (> day 29). The glycogen concen a ions in he e e - ence cul i a ions (0% NaCl) ob ained 12.45 ± 0.66%CDW (PCC6803), 23.04 ± 1.21% CDW (CCALA192) and 12.80 ± 0.48% CDW (IFA3) on day 21. By inc easing salini y o 6%, glycogen concen a ions o 20.67 ± 1.25% CDW (PCC6803), 20.93 ± 0.63% CDW (CCALA192) and 20.26 ± 1.48% CDW (IFA3) we e achie ed on day 21, while on day 42 i had jus inc eased owa d 27.24 ± 1.18% CDW, 33.46 ± 2.63% CDW and 35.05 ± 1.66% CDW in PCC6803, CCALA192 and IFA3, espec i ely. In con as o PHB, glycogen le els we e high a low sal concen a ions. PHB and glycogen in common inc eased Fig. 3 PCC6803 (a) g ow h, (b) PHB ( illed columns) and glycogen (dashed columns), (c) chlo ophylla ( illed columns) and phycocyanin (dashed columns) as well as (d) ca o enoids in 0%, 3%, 6% and 8% NaCl added o he medium; n = 3, e o ba s = s anda d de ia ion 1232 Jou nal o Applied Phycology (2022) 34:1227–1241 1 3 up o ce ain sal concen a ions (e.g., 3% NaCl o glyco- gen and 4% NaCl o PHB, bo h in CCALA192) and hen declined. Fo glycogen, highe concen a ions a e s a ed o be p oduced in 4% NaCl han in 0% (Pade e al. 2017). When compa ing e e ence cul i a ions (0% NaCl) o Synechocys- is PCC6803 highe glycogen alues we e achie ed in he li e a u e—24.1% CDW wi hin 21days (Velmu ugan and Incha oensakdi 2018) — han compa ed o he ein cul i a ed PCC6803 and IFA3 (12.45 ± 0.66%CDW and 12.80 ± 0.48% CDW, espec i ely, in e e ence cul i a ions (0% NaCl), day 21). The alues ob ained by CCALA192 (23.04 ± 1.21% CDW, e e ence cul i a ions (0% NaCl), day 21) we e wi hin he ange. By gene ic modi ica ions (glycogen o e exp ession combined wi h phaA-knockou ), glycogen concen a ions we e u he inc eased o 38.8% CDWon day 20 and 40.4% CDWon day 25 (Velmu ugan and Incha oensakdi 2018). These alues we e wice as high as hose achie ed in he e - e ence cul i a ions (0% NaCl) on day 21 bu wi hin he ange ob ained a 3% NaCl a e 42 cul i a ion days (PCC6803: 45.84 ± 2.02% CDW; CCALA192: 49.43 ± 0.58% CDW; IFA3: 42.85 ± 1.08% CDW). Glycogen concen a ions can be e en u he enhanced by ni ogen dep i a ion ( o abou 42% CDW, a e 12days). Besides ha , glycogen concen a ions especially a he beginning o cul i a ion a e ele a ed when cells a e p e-g own unde pho omixo ophic condi ions (sligh ly below 60% CDW a he beginning o cul i a ion and sligh ly abo e 50% CDW a e 12days) compa ed o pho o o- phic condi ions (abou 3% CDW a he beginning and sligh ly o e 40% CDW a e 12days) (Du and S i as a a 2018). Sal S ess andi s E ec on heAccumula ion o PHB andGlycogen The a e age PHB- o-glycogen a ios o e he cul i a ion ime in 3% sal we e compa able be ween all h ee s ains (1:21 o CCALA192, 1:24 o PCC6803 and 1:27 o IFA3), sugges ing ha PHB and glycogen syn hesis pa hways in all h ee s ains we e abou equally s ong a mode a e sal concen a ions. Wi h inc easing o dec easing sal concen a ions his a io changed. PCC6803 p oduced 40 and 147 imes mo e and IFA3 55 and 129 imes mo e glycogen han PHB a 0% and 6% NaCl, espec i ely. Only glycogen accumula ion in CCALA192 s ood a he cons an a 1:25 and 1:29 in 0% and 6% NaCl. Summa ized, all h ee s ains accumula ed mo e glycogen han PHB, bu a ios changed wi h changing salini y. The ac ha medium Fig. 4 CCALA192 (a) g ow h, (b) PHB ( illed columns) and glycogen (dashed columns), (c) chlo ophylla ( illed columns) and phycocyanin (dashed columns) as well as (d) ca o enoids in 0%, 3%, 6% and 8% NaCl added o he medium; n = 3, e o ba s = s anda d de ia ion 1233Jou nal o Applied Phycology (2022) 34:1227–1241 1 3 salini y subs an ially a ec s PHB and glycogen con en in cyanobac e ia is no only o undamen al in e es bu i migh be also o bio echnological impo ance since NaCl addi ion could be used as a ool o enhance PHB syn hesis; ne e heless, he sal should be applied a la e s ages o cul i a ion o no inhibi he g ow h o he cul u e (since PHB is an in acellula me aboli e and high CDW alues a e he necessa y p e equisi e o high PHB i e s). The ac ha ac ions o bo h s o age compounds a e lowe in cul u es exposed o high osmo ic p essu es can be a ibu ed o inhibi ion o hepho osyn he ic sys em esul - ing in a dec eased amoun o ca bon and ene gy o s o age polyme s syn hesis (see u he in he ex ). Ne e heless, i is also likely ha ca bon and ene gy expenses o s ess- esponse agains osmo ic p essu e (exc e ion o Na+, p oduc- ion o compa ible solu es, modula ion o gene exp ession e c.) also con ibu ed o dec eased amoun s o s o age poly- me s in he cul u es. Hence, i NaCl should be conside ed as a ool o imp o e PHB p oduc ion, i s concen a ion mus be wisely chosen and p ecisely con olled since abo e ace ain aluei has a nega i e impac on PHB syn hesis. Spec oscopic Iden i ica ion o Pigmen s inCell Suspensionso Synechocys is s ains PCC6803 andCCALA192 To iden i y and analyze pigmen s di ec ly in he cul i a ed cul- u e, we used a less common spec oscopic me hod o di usi e ansmission spec opho ome y ha allows he de e mina ion o UV-VIS abso p ion spec a o highly u bid samples. All he measu ed cul u es we e a he same s age o cul i a ion (42days). As can be seen om he compa ison o co esponding pigmen con en s shown in Fig.3c/d, Fig.4c/d, Fig.5c/d and changes in he spec al inge p in s o he pigmen s in Fig.6, he e is a good ag eemen in he obse ed e ec o he sal p esen in cul i a ion media on he changes in pigmen s composi ion. Quan i ica ion o Pigmen sob ained byEx ac ion Chlo ophylla In all he h ee Synechocys is s ains, he chlo ophylla con en s ose un il day 14 (0% and 3% NaCl, PCC6803, CCALA192, IFA3) o 21 (6% NaCl, PCC6803, CCALA192, Fig. 5 IFA3 (a) g ow h, (b) PHB ( illed columns) and glycogen (dashed columns), (c) chlo ophylla ( illed columns) and phycocyanin (dashed columns) as well as (d) ca o enoids in 0%, 3%, 6% and 8% NaCl added o he medium; n = 3, e o ba s = s anda d de ia ion 1234 Jou nal o Applied Phycology (2022) 34:1227–1241 1 3 IFA3) and hen dec eased wi h inc easing cul i a ion ime. Addi ionally, chlo ophylla ends o dec ease a highe sal s ess le els (PCC6803: Fig.3c, CCALA192: Fig.4c, IFA3: Fig.5c). Chlo ophylla was highes in PCC6803 (1.34 ± 0.02% CDW) in 2% NaCl on day 14, compa ed o IFA3 and CCALA192, whose chlo ophylla le els also peaked (1.01 ± 0.03% CDW and 0.93 ± 0.06% CDW, espec- i ely) in 2% NaCl on day 14. Conce ning cul i a ion ime, an inc easing end o chlo opylla in Synechocys is PCC6803 and enginee ed s ains (glycogen o e exp ession, phaA-knockou ) he eo is epo ed o g ow h in sal ee BG-11 medium om day 10 o 15, ollowed by a dec ease o day 20 (Velmu ugan and Incha oensakdi 2018). This end could be con i med by PCC6803, CCALA192 and IFA3 whose chlo ophylla con en in e e ence cul i a ions (0% NaCl) ose un il day 14 o 21 and hen declined un il he end o cul i a ion. The esul s ob ained o chlo ophylla a di e en sal concen a ions we e compa able wi h hose collec ed by Sudhi and Mu hy (2004), who s a ed ha a 342mM (2%) NaCl he chlo ophylla con en in Synechocys is PCC6803 inc eased, while a 684mM (4%) o 1026mM (6%) i sha ply dec eased. As he chlo ophylla con en dec eased, g ow h also dec eased bu did no s op comple ely. The dec ease in chlo ophyllawas no only obse ed wi h cul i a ion ime, bu also wi h inc easing sal concen a ion. Highe sal concen a ions led o a as e decline in chlo ophylla. How- e e , e en low chlo ophylla concen a ions we e su icien o Synechocys is o con inue g owing. In case o ni ogen chlo osis, phycocyanin (peak a 630nm) and o a lesse ex en chlo ophylla (peak a 680nm) g adually dec eased wi h inc easing cul i a ion ime (K asiko e al. 2012) and g ow h s opped (Klo z e al. 2016). In glycogen o e exp essing s ains, chlo opylla con en s we e lowe compa ed o he wild ype and phaA-knockou s ain, espec i ely. Despi e low chlo ophylla con en s, oxy- gen e olu ion a e was highes in he combined s ain (gly- cogen o e exp ession and phaA-knockou ), which enhanced o e all biomass concen a ion (Velmu ugan and Incha oen- sakdi 2018). Simila pa e ns can be seen when compa - ing chlo ophylla and glycogen con en s o PCC6803 and CCALA192. Chlo ophylla con en s o PCC6803 we e gen- e ally highe , while glycogen le els and g ow h we e sligh ly lowe han in CCALA192. This sugges s ha he e iciency o he con en o PSII (pho osys em II, o which oxygen e o- lu ion is connec ed (Allakh e die and Mu a a 2008)) in PCC6803 is a he low compa ed o he o he wo s ains. Phycocyanin Phycocyanin showed simila endencies as chlo opylla; i s con en s a he dec eased wi h inc easing cul i a ion ime (PCC6803: om day 7 a 3%, om day 29 a 6%; CCALA192: om day 7 a 0%, om day 14 a 3%, om day 21 a 6%; IFA3: om day 7 a 0% and 3%, om day 21 a 6%) (PCC6803: Fig.3c, CCALA192: Fig.4c, IFA3: Fig.5c). Rela ed o sal concen a ions, he pic u e is mo e complex: The highes phycocyanin le els we e eached by CCALA192 (9.17 ± 1.38% CDW) in 3% NaCl a day 14 and by IFA3 (7.17% CDW) in 0% a day 7. PCC6803 eached a maximum o only 2.34 ± 0.05% CDW in 3% NaCl on day 7. In 4% NaCl phycocyanin concen a ions we e com- pa ably low— he maximum was 2.32 ± 0.11% CDW in CCALA192 a day 14. A highe salini ies, phycocyanin Fig. 6 VIS abso p ion spec a (400–750nm) o cul u es a e 42days o cul i a ion in 0%, 3%, 6% and 8% NaCl added in o he medium a) PCC6803 b) CCALA192. Spec a we e ob ained by di usi e ansmis- sion spec opho ome y 1235Jou nal o Applied Phycology (2022) 34:1227–1241