Vol.:(0123456789)
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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