wa e
A icle
Remo al o Mic ocys is ae uginosa h ough he
Combined E ec o Plasma Discha ge and
Hyd odynamic Ca i a ion
Blahosla Ma šálek 1,*, Eliška Ma šálko á1, Klá a Odehnalo á1, F an išek Pochylý2,
Pa el Rudol 2, Pa el S ahel 3, Joze Rahel 3, Jan ˇ
Cech 3, Simona Fialo á2and
Š ˇepán Zezulka 1
1Ins i u e o Bo any, Czech Academy o Sciences, Lidická25/27, CZ-602 00 B no, Czech Republic;
[email p o ec ed] (E.M.); [email p o ec ed] (K.O.); [email p o ec ed] (Š.Z.)
2V. Kaplan Depa men , Facul y o Mechanical Enginee ing, B no Uni e si y o Technology, 601 90 B no,
Czech Republic; [email p o ec ed].cz (F.P.); [email p o ec ed].cz (P.R.); [email p o ec ed].cz (S.F.)
3Depa men o Physical Elec onics, Facul y o Science, Masa yk Uni e si y, Ko lᡠská2, CZ-611 37 B no,
Czech Republic; [email p o ec ed] (P.S.); [email p o ec ed] (J.R.); [email p o ec ed] (J. ˇ
C.)
*Co espondence: blahosla [email p o ec ed]; Tel.: +42-0603-8729-55
Recei ed: 19 No embe 2019; Accep ed: 13 Decembe 2019; Published: 18 Decembe 2019


Abs ac :
Cyanobac e ial wa e blooms ep esen oxicological, ecological and echnological p oblems
a ound he globe. When p esen in aw wa e used o d inking wa e p oduc ion, one o he bes
s a egies is o emo e he cyanobac e ial biomass gen ly be o e ea men , a oiding cell des uc ion
and cyano oxins elease. This pape p esen s a new me hod o he emo al o cyanobac e ial
biomass du ing d inking wa e p e- ea men ha combines hyd odynamic ca i a ion wi h cold
plasma discha ge. Ca i a ion p oduces p ess s ess ha causes Mic ocys is gas esicles o collapse.
The cyanobac e ia hen sink, allowing o emo al by sedimen a ion. The cyanobac e ia showed no
signs o e i alisa ion, e en a e se en days unde op imal condi ions wi h nu ien en ichmen ,
as pho osyn he ic ac i i y is nega i ely a ec ed by hyd ogen pe oxide p oduced by plasma bu n in
he ca i a ion cloud. Using his me hod, cyanobac e ia can be emo ed in a single ea men , wi h no
inc ease in mic ocys in concen a ion. This no el echnology appea s o be highly p omising o
con inual ea men o aw wa e in low in d inking wa e ea men plan s and will also be o in e es
o hose wishing o ea su ace wa e s wi hou he use o algaecides.
Keywo ds: cyanobac e ial bloom; wa e ea men ; d inking wa e ; su ace wa e ; cold plasma
1. In oduc ion
Inc easing eu ophica ion o su ace wa e s esul s in he de elopmen o cyanobac e ial blooms,
esul ing in a de e io a ion o wa e quali y and dis up ions in he use o wa e bodies o ec ea ional,
echnological and ishe ies pu poses o d inking wa e p oduc ion [
1
]. Fu he mo e, he e is clea
e idence indica ing ha cyanobac e ial blooms a e inc easing in equency, magni ude and du a ion
globally and ha hey a e becoming a majo echnological, oxicological and hygienic p oblem [1].
Va ious me hods a e a ailable o educing o emo ing cyanobac e ial blooms. A b oad spec um
o chemical compounds can be used as p e en i e o cu a i e measu es [
2
]; howe e , hese can
cause addi ional eco oxicological isks o aqua ic ecosys ems [
3
]. Fu he s a egies o p e en ing
he de elopmen o cyanobac e ial blooms a e nu ien limi a ion [
4
], semi-na u al me hods such as
he use o plan ex ac s [
5
,
6
] o ba ley s aw, and biological me hods such as he use o p obio ic
mic oo ganisms and bio ic in e ac ions [
7
]. O he many chemical and biological me hods a ailable,
physical me hods a e being inc easingly in es iga ed, as hey no only emo e cyanobac e ial biomass
Wa e 2020,12, 8; doi:10.3390/w12010008 www.mdpi.com/jou nal/wa e
Wa e 2020,12, 8 2 o 14
bu also cyano oxins, which is especially impo an o d inking wa e ea men . While elec ochemical
me hods, such as elec opo a ion, elec ocoagula ion o elec o lo a ion [
8
], ha e been he subjec o
pa icula in e es , plasma discha ge in combina ion wi h ad anced oxida ion echnologies o i ied by
ul asonic o hyd odynamic ca i a ion a e p obably he mos s udied me hods a his ime [9–11].
In a s udy Kim e al. [
12
] ha e alua ed he educ ion o algal biomass by applying non- he mal o
cold-plasma p ocesses, i was ound ha such me hods des oyed he cell walls o mic oalgae. Based
on hei esea ch, cold plasma seems o be a use ul op ion o e ec i ely ea ing pollu ion caused by
algal blooms in su ace wa e . Plasma and ca i a ion echnologies ha e been applied o pu i ying
wa e con amina ed by mic oo ganisms [
10
,
13
]; howe e , p ac ical applica ions ha e been se e ely
es ic ed by excessi e ea men imes and ene gy demand (e.g., 24 h o cold plasma ea men o
se e al hund ed ca i a ion cycles).
As such, he e is a clea need o echnologies ha can emo e cyanobac e ial biomass wi hou
eleasing cyano oxins, and ha can be applied in con inuous mode o d inking wa e p e- ea men .
In his s udy, we ocus on physical me hods ha combine hyd odynamic ca i a ion (HC) and plasma
discha ge (HC +plasma). In p inciple, he me hod elies on he o ma ion o apou ca i ies in a liquid,
wi h plasma being p oduced in he bubble cloud ha is o med downs eam o he eac o nozzle
h ough ca i a ion. Plasma is p oduced h ough he applica ion o high ol age o an elec ode pai
moun ed in he eac o [
14
]. We modi ied his me hod o gen le ea men o cyanobac e ial biomass
wi h he aim o educing cyanobac e ial cell pho osyn he ic ac i i y wi hou cell lysis o elease o
cyano oxins. By op imising he ime, ene ge ic and ea men aspec s o he me hod, we aim o de elop
a p ac ically applicable me hod o use in d inking wa e ea men plan s.
2. Ma e ials and Me hods
2.1. Hyd odynamic Ca i a ion De ice
We cons uc ed an expe imen al ci cui consis ing o a wa e ank, a cen i ugal pump (Calpeda,
Vicenza, I aly, a ed powe ou pu 0.1 kW) con olled by a equency con e o , a se o p essu e
ansduce s (p
1
and p
2
; bo h BD Senso , 0–6 ba ) and a lowme e (Flomag DN 15) (Figu e 1).
A con e ging–di e ging (CD) nozzle wi h an inle diame e o 10 mm and a minimum diame e o
4 mm a he h oa sec ion was manu ac u ed om plexiglass. We designed a nozzle ha p oduced
a ca i a ion egion illed wi h sa u a ed apou ollowing a p essu e d op om c oss-sec ional a ea
educ ion (i.e., es ablishmen o HC acco ding o he Be noulli p inciple). As apou p essu e is
sensi i e o wa e empe a u e and he amoun o dissol ed gas, a he mome e (Sensi , ype PT100)
and p obe measu ing dissol ed oxygen con en (Oxymax) we e inco po a ed in o he expe imen al
ci cui . Subsequen moni o ing con i med ha dissol ed oxygen emained a ca. 8.3 mg/L h oughou
he expe imen , ha s a ic p essu e a he nozzle h oa eached sa u a ed apou p essu e le els a
3500 Pa and ha liquid empe a u e emained ela i ely s able o he i s i e cycles o HC +plasma
ea men , a ying only sligh ly be ween 23 and 25
◦
C. All es s we e ca ied ou a a discha ge a e o
0.45 L/s.
Wa e 2019, 11, x FOR PEER REVIEW 2 o 17
cyanobac e ial biomass bu also cyano oxins, which is especially impo an o d inking wa e
ea men . While elec ochemical me hods, such as elec opo a ion, elec ocoagula ion o
elec o lo a ion [8], ha e been he subjec o pa icula in e es , plasma discha ge in combina ion wi h
ad anced oxida ion echnologies o i ied by ul asonic o hyd odynamic ca i a ion a e p obably he
mos s udied me hods a his ime [9–11].
In a s udy Kim e al. [12] ha e alua ed he educ ion o algal biomass by applying non- he mal
o cold-plasma p ocesses, i was ound ha such me hods des oyed he cell walls o mic oalgae.
Based on hei esea ch, cold plasma seems o be a use ul op ion o e ec i ely ea ing pollu ion
caused by algal blooms in su ace wa e . Plasma and ca i a ion echnologies ha e been applied o
pu i ying wa e con amina ed by mic oo ganisms [10,13]; howe e , p ac ical applica ions ha e been
se e ely es ic ed by excessi e ea men imes and ene gy demand (e.g., 24 h o cold plasma
ea men o se e al hund ed ca i a ion cycles).
As such, he e is a clea need o echnologies ha can emo e cyanobac e ial biomass wi hou
eleasing cyano oxins, and ha can be applied in con inuous mode o d inking wa e p e- ea men .
In his s udy, we ocus on physical me hods ha combine hyd odynamic ca i a ion (HC) and plasma
discha ge (HC + plasma). In p inciple, he me hod elies on he o ma ion o apou ca i ies in a
liquid, wi h plasma being p oduced in he bubble cloud ha is o med downs eam o he eac o
nozzle h ough ca i a ion. Plasma is p oduced h ough he applica ion o high ol age o an elec ode
pai moun ed in he eac o [14]. We modi ied his me hod o gen le ea men o cyanobac e ial
biomass wi h he aim o educing cyanobac e ial cell pho osyn he ic ac i i y wi hou cell lysis o
elease o cyano oxins. By op imising he ime, ene ge ic and ea men aspec s o he me hod, we
aim o de elop a p ac ically applicable me hod o use in d inking wa e ea men plan s.
2. Ma e ials and Me hods
2.1. Hyd odynamic Ca i a ion De ice
We cons uc ed an expe imen al ci cui consis ing o a wa e ank, a cen i ugal pump (Calpeda,
Vicenza, I aly, a ed powe ou pu 0.1 kW) con olled by a equency con e o , a se o p essu e
ansduce s (p
1
and p
2
; bo h BD Senso , 0–6 ba ) and a lowme e (Flomag DN 15) (Figu e 1). A
con e ging–di e ging (CD) nozzle wi h an inle diame e o 10 mm and a minimum diame e o 4
mm a he h oa sec ion was manu ac u ed om plexiglass. We designed a nozzle ha p oduced a
ca i a ion egion illed wi h sa u a ed apou ollowing a p essu e d op om c oss-sec ional a ea
educ ion (i.e., es ablishmen o HC acco ding o he Be noulli p inciple). As apou p essu e is
sensi i e o wa e empe a u e and he amoun o dissol ed gas, a he mome e (Sensi , ype PT100)
and p obe measu ing dissol ed oxygen con en (Oxymax) we e inco po a ed in o he expe imen al
ci cui . Subsequen moni o ing con i med ha dissol ed oxygen emained a ca. 8.3 mg/L h oughou
he expe imen , ha s a ic p essu e a he nozzle h oa eached sa u a ed apou p essu e le els a
3500 Pa and ha liquid empe a u e emained ela i ely s able o he i s i e cycles o HC + plasma
ea men , a ying only sligh ly be ween 23 and 25 °C. All es s we e ca ied ou a a discha ge a e
o 0.45 L/s.
Figu e 1. Schema ic illus a ion o he hyd odynamic ca i a ion (HC) de ice [15]. CD: con e ging–
di e ging.
Figu e 1.
Schema ic illus a ion o hehyd odynamicca i a ion (HC)de ice[
15
]. CD: con e ging–di e ging.
Wa e 2020,12, 8 3 o 14
2.2. Plasma Je Gene a ion in Liquid Gas Ca i ies
A plasma je was p oduced in he ca i a ion bubble cloud gene a ed in wa e using an expe imen al
se up consis ing o wo pa s: (a) a PMMA (poly me hyl me hac yla e, i.e., plexiglass) pipe ca i a ion
gene a o (nozzle) connec ed o a wa e pumping and eci cula ion uni (black ci cle in Figu e 2),
and (b) a plasma gene a o wi h a discha ge elec ode sys em and a high- ol age (HV) powe gene a o
(g een ci cle in Figu e 2). Spec al analysis o he discha ge was ob ained jus abo e he HV elec ode
using A aSpec ULS3648TEC-USB2 spec ome e s (A an es BV, The Ne he lands). Spec ome e s we e
con igu ed o h ee spec al egions co e ing he ange om nea -ul a iole (UV) o he nea -in a ed
(NIR) egion. In he con igu a ion o su ey spec a (UV-VIS-NIR), he spec ome e was equipped
wi h he g a ing UA (200–1100 nm), a sli o 25 um, Deep UV de ec o coa ing and o de -so ing coa ing
wi h 350 and 600 nm long-pass il e . Fo highe esolu ion measu emen s in UV egion, esp. NIR
egion, he spec ome e s we e equipped wi h he g a ing UE (290–395 nm), sli 10 um and DUV
coa ing, esp. g a ing NC (740–924 nm), sli 25 um and o de -so ing il e a 600 nm.
Wa e 2019, 11, x FOR PEER REVIEW 3 o 17
2.2. Plasma Je Gene a ion in Liquid Gas Ca i ies
A plasma je was p oduced in he ca i a ion bubble cloud gene a ed in wa e using an
expe imen al se up consis ing o wo pa s: (a) a PMMA (poly me hyl me hac yla e, i.e., plexiglass)
pipe ca i a ion gene a o (nozzle) connec ed o a wa e pumping and eci cula ion uni (black ci cle
in Figu e 2), and (b) a plasma gene a o wi h a discha ge elec ode sys em and a high- ol age (HV)
powe gene a o (g een ci cle in Figu e 2). Spec al analysis o he discha ge was ob ained jus abo e
he HV elec ode using A aSpec ULS3648TEC-USB2 spec ome e s (A an es BV, The Ne he lands).
Spec ome e s we e con igu ed o h ee spec al egions co e ing he ange om nea -ul a iole
(UV) o he nea -in a ed (NIR) egion. In he con igu a ion o su ey spec a (UV-VIS-NIR), he
spec ome e was equipped wi h he g a ing UA (200–1100 nm), a sli o 25 um, Deep UV de ec o
coa ing and o de -so ing coa ing wi h 350 and 600 nm long-pass il e . Fo highe esolu ion
measu emen s in UV egion, esp. NIR egion, he spec ome e s we e equipped wi h he g a ing UE
(290–395 nm), sli 10 um and DUV coa ing, esp. g a ing NC (740–924 nm), sli 25 um and o de -
so ing il e a 600 nm.
Figu e 2. Expe imen al se -up.
We gene a ed a plasma je in he ca i a ion egion using a pai o PVC-insula ed coppe wi e
elec odes placed inside he ca i a ion nozzle (Figu e 3). The i s HV elec ode was placed inside he
nozzle a he poin o minimum diame e , while he second, g ounded, elec ode was posi ioned
downs eam o he ca i a ion bubble cloud. The insula ion was s ipped om he ips o he wi es o
c ea e a di ec conduc ing con ac wi h he wa e /bubble en i onmen (see Figu e 3). The discha ge
was gene a ed using an HV gene a o wi h sinusoidal HV ou pu , wi h ope a ion equency se o 50
kHz and HV powe a 400 W. The plasma je was gene a ed be ween he ips o he HV elec ode and
he g ounded elec ode, whe e i o med a plasma a ea ca. 20 mm in leng h (Figu e 4). To al wa e
olume inside he sys em was 6 L, and con ac - ea men ime was 1.48 s.
Figu e 3. De ails o he elec ode sys em and i s placemen wi h espec o he ca i a ion cloud.
Figu e 2. Expe imen al se -up.
We gene a ed a plasma je in he ca i a ion egion using a pai o PVC-insula ed coppe wi e
elec odes placed inside he ca i a ion nozzle (Figu e 3). The i s HV elec ode was placed inside
he nozzle a he poin o minimum diame e , while he second, g ounded, elec ode was posi ioned
downs eam o he ca i a ion bubble cloud. The insula ion was s ipped om he ips o he wi es o
c ea e a di ec conduc ing con ac wi h he wa e /bubble en i onmen (see Figu e 3). The discha ge
was gene a ed using an HV gene a o wi h sinusoidal HV ou pu , wi h ope a ion equency se o
50 kHz and HV powe a 400 W. The plasma je was gene a ed be ween he ips o he HV elec ode
and he g ounded elec ode, whe e i o med a plasma a ea ca. 20 mm in leng h (Figu e 4). To al wa e
olume inside he sys em was 6 L, and con ac - ea men ime was 1.48 s.
Wa e 2019, 11, x FOR PEER REVIEW 3 o 17
2.2. Plasma Je Gene a ion in Liquid Gas Ca i ies
A plasma je was p oduced in he ca i a ion bubble cloud gene a ed in wa e using an
expe imen al se up consis ing o wo pa s: (a) a PMMA (poly me hyl me hac yla e, i.e., plexiglass)
pipe ca i a ion gene a o (nozzle) connec ed o a wa e pumping and eci cula ion uni (black ci cle
in Figu e 2), and (b) a plasma gene a o wi h a discha ge elec ode sys em and a high- ol age (HV)
powe gene a o (g een ci cle in Figu e 2). Spec al analysis o he discha ge was ob ained jus abo e
he HV elec ode using A aSpec ULS3648TEC-USB2 spec ome e s (A an es BV, The Ne he lands).
Spec ome e s we e con igu ed o h ee spec al egions co e ing he ange om nea -ul a iole
(UV) o he nea -in a ed (NIR) egion. In he con igu a ion o su ey spec a (UV-VIS-NIR), he
spec ome e was equipped wi h he g a ing UA (200–1100 nm), a sli o 25 um, Deep UV de ec o
coa ing and o de -so ing coa ing wi h 350 and 600 nm long-pass il e . Fo highe esolu ion
measu emen s in UV egion, esp. NIR egion, he spec ome e s we e equipped wi h he g a ing UE
(290–395 nm), sli 10 um and DUV coa ing, esp. g a ing NC (740–924 nm), sli 25 um and o de -
so ing il e a 600 nm.
Figu e 2. Expe imen al se -up.
We gene a ed a plasma je in he ca i a ion egion using a pai o PVC-insula ed coppe wi e
elec odes placed inside he ca i a ion nozzle (Figu e 3). The i s HV elec ode was placed inside he
nozzle a he poin o minimum diame e , while he second, g ounded, elec ode was posi ioned
downs eam o he ca i a ion bubble cloud. The insula ion was s ipped om he ips o he wi es o
c ea e a di ec conduc ing con ac wi h he wa e /bubble en i onmen (see Figu e 3). The discha ge
was gene a ed using an HV gene a o wi h sinusoidal HV ou pu , wi h ope a ion equency se o 50
kHz and HV powe a 400 W. The plasma je was gene a ed be ween he ips o he HV elec ode and
he g ounded elec ode, whe e i o med a plasma a ea ca. 20 mm in leng h (Figu e 4). To al wa e
olume inside he sys em was 6 L, and con ac - ea men ime was 1.48 s.
Figu e 3. De ails o he elec ode sys em and i s placemen wi h espec o he ca i a ion cloud.
Figu e 3. De ails o he elec ode sys em and i s placemen wi h espec o he ca i a ion cloud.
2.3. Cyanobac e ial S ain and Cul u e Condi ions
Fo ou expe imen s, we used Mic ocys is ae uginosa PCC 7806 s ain (Pas eu Cul u e Collec ion
o Cyanobac e ia; Pa is) cul u ed in BG11 medium o cyanobac e ia ( o medium composi ion,
see Supplemen a y Ma e ials Table S1). The cul u e has now been placed in ou labo a o y cul u e
collec ion and is g own unde s anda d condi ions o cyanobac e ia.
Wa e 2020,12, 8 4 o 14
Wa e 2019, 11, x FOR PEER REVIEW 4 o 17
Figu e 4. Image o he plasma je gene a ed in he liquid/ca i a ion bubble cloud en i onmen .
2.3. Cyanobac e ial S ain and Cul u e Condi ions
Fo ou expe imen s, we used Mic ocys is ae uginosa PCC 7806 s ain (Pas eu Cul u e Collec ion
o Cyanobac e ia; Pa is) cul u ed in BG11 medium o cyanobac e ia ( o medium composi ion, see
Supplemen a y Ma e ials Table S1). The cul u e has now been placed in ou labo a o y cul u e
collec ion and is g own unde s anda d condi ions o cyanobac e ia.
2.4. De e mina ion o Hyd ogen Pe oxide
We de e mined hyd ogen pe oxide p oduc ion using a colo ime ic me hod based on he
eac ion o pe oxide wi h i anyl ions ha p oduces a yellow-colou ed complex o pe i anic acid:
Ti4+ + H2O2 + H2O → TiO2.H2O2 + 4H+ [16]
2.5. G ow h Inhibi ion Tes
Immedia ely a e each expe imen , a cul u e o he Mic ocys is biomass was assessed using a
modi ied e sion o s anda d me hod ISO 8692 [17,18]. Samples om he expe imen al ea men (HC
o HC + Plasma) we e mixed and homogenised, ollowing which 1 o 2 mL o sample was mixed wi h
50% g ow h medium a a a io o 1:1 in o de o p o ide op imal g ow h condi ions. The samples
we e hen pipe ed in o a 96-well pla e and chlo ophyll luo escence was measu ed each 24 h using
a Tecan SPARK mul i unc ional mic opla e eade (Aus ia).
2.6. Chlo ophyll Quan i ica ion
Chlo ophyll concen a ion was assessed using a bbe Moldaenke Fluo oP obe (Ge many), which
bo h de e mines and quan i ies he algae classed unde g een algae, cyanobac e ia, dia oms and
c yp ophy es. Samples we e ob ained om 2 cm below he essel su ace.
2.7. Measu emen o Pho osyn he ic Ac i i y
Induced chlo ophyll luo escence (ChlFl) cha ac e is ics we e assessed using he AquaPen AP
100-C luo ome e (PSI, D áso , Czech Republic) equipped wi h cu e e holde and ambe - ed (620
nm) LEDs as measu ing, ac inic and sa u a ion ligh sou ces. A se o ChlFl pa ame e s was achie ed
a e 10 min o da k p e-adap a ion a oom empe a u e (22 ± 1 °C). Immedia ely be o e he
measu emen , samples in he cu e e we e ho oughly shaken o a oid sedimen a ion and/o su ace
loa ing o cyanobac e ial cells, hen a sa u a ion pulse (3000 µmol m−2 s−1, 500 ms) was applied and
he maximal quan um yield (FV/FM) was measu ed. A e ano he 30 s o elaxa ion in he da k, as
Commen ed [M1]: 补补补补补补
Figu e 4. Image o he plasma je gene a ed in he liquid/ca i a ion bubble cloud en i onmen .
2.4. De e mina ion o Hyd ogen Pe oxide
We de e mined hyd ogen pe oxide p oduc ion using a colo ime ic me hod based on he eac ion
o pe oxide wi h i anyl ions ha p oduces a yellow-colou ed complex o pe i anic acid [16]:
Ti4++H2O2+H2O→TiO2.H2O2+4H+
2.5. G ow h Inhibi ion Tes
Immedia ely a e each expe imen , a cul u e o he Mic ocys is biomass was assessed using a
modi ied e sion o s anda d me hod ISO 8692 [
17
,
18
]. Samples om he expe imen al ea men (HC
o HC +Plasma) we e mixed and homogenised, ollowing which 1 o 2 mL o sample was mixed wi h
50% g ow h medium a a a io o 1:1 in o de o p o ide op imal g ow h condi ions. The samples we e
hen pipe ed in o a 96-well pla e and chlo ophyll luo escence was measu ed each 24 h using a Tecan
SPARK mul i unc ional mic opla e eade (Aus ia).
2.6. Chlo ophyll Quan i ica ion
Chlo ophyll concen a ion was assessed using a bbe Moldaenke Fluo oP obe (Ge many), which
bo h de e mines and quan i ies he algae classed unde g een algae, cyanobac e ia, dia oms and
c yp ophy es. Samples we e ob ained om 2 cm below he essel su ace.
2.7. Measu emen o Pho osyn he ic Ac i i y
Induced chlo ophyll luo escence (ChlFl) cha ac e is ics we e assessed using he AquaPen AP
100-C luo ome e (PSI, D
á
so , Czech Republic) equipped wi h cu e e holde and ambe - ed (620 nm)
LEDs as measu ing, ac inic and sa u a ion ligh sou ces. A se o ChlFl pa ame e s was achie ed a e
10 min o da k p e-adap a ion a oom empe a u e (22
±
1
◦
C). Immedia ely be o e he measu emen ,
samples in he cu e e we e ho oughly shaken o a oid sedimen a ion and/o su ace loa ing o
cyanobac e ial cells, hen a sa u a ion pulse (3000
µ
mol m
−2
s
−1
, 500 ms) was applied and he maximal
quan um yield (F
V
/F
M
) was measu ed. A e ano he 30 s o elaxa ion in he da k, as luo escence
ansien (OJIP cu e) unde ac inic ligh (300
µ
mol m
−2
s
−1
, 2 s) was eco ded. Th ee independen
samples om each ea men we e analysed and ChlFl pa ame e s including basal and maximal
luo escence le el (F
0
, F
M
o F
P
) o ‘Fix A ea’ alue we e eco ded. In o de o compa e he i ness
o cyanobac e ial cells ela i ely and independen ly om he F
V
/F
M
alue, a “Fi ness ac o ” was
in oduced as a a io o he Fix A ea alue (de ined ma hema ically as he a ea below he OJIP cu e
limi ed by F
0
ime o es ima ion (40
µ
s) and ime 1 s, a e he ex ac ion o backg ound signal le el) o
F0acco ding o he ollowing equa ion:
Fi ness ac o =(Fix A ea/F0)−1000 (uni less)
Wa e 2020,12, 8 5 o 14
This alue in eg a es bo h he o e all luo escence signal based on cell suspension p ope ies
(densi y, chlo ophyll con en ) and changes in he shape o he OJIP cu e.
2.8. Mic ocys in Analysis
Quan iPla e Ki o Mic ocys ins (En i oLogix USA) was used o ex acellula mic ocys ins
de e mina ion. A e expe imen al ea men s, samples we e il e ed by 0.2
µ
m Sa o ius il e s. Resul s
a e in e p e ed as he sum o mic ocys ins.
2.9. S a is ical Analysis
To ind ou i he da ase s o he expe imen al esul s we e s a is ically signi ican , analysis o
a iance (ANOVA) was used.
A e e i ica ion o no mal dis ibu ion and homogenei y o a iance, he da a we e p ocessed by
analysis o a iance (ANOVA), wi h di e ences compa ed he Tukey Hones ly signi ican di e ence
(HSD) ange es and signi icance se a p<0.05.
3. Resul s and Discussion
3.1. Plasma Gene a ion
While ac i i y o eac i e oxygen species (ROS) has been p oposed as he d i e o cyanobac e ial
cell damage in p e ious s udies epo ing on he use o plasma in wa e ea men , he e is no empi ical
da a o suppo his. In his s udy, we speci ically se ou o measu e he p esence o adicals gene a ed.
In doing so, we no only con i med he p esence o oxygen adicals [OH] bu also an app eciable le el
o ni ogen adicals (see Figu e 5). Spec al analysis o he discha ge ( aken jus abo e he HV elec ode;
spec al lines and bands iden i ied using [
19
–
21
]; see Figu e 5, o de ails see Supplemen a y Ma e ials)
unde bo h nea UV (Figu e 6) and NIR (Figu e 7) clea ly showed ha he combina ion o HC +plasma
p oduced bo h oxygen adicals (single and/o iple spec a) and hyd oxyl o ni oxyl adicals. While
he wa e was only exposed o plasma o ca. 7 ms pe cycle, he eac i e oxygen/ni ogen species
(RONS) gene a ed (e.g., pe oxides) ha e a longe li e ime. The spec a also included a omic coppe
lines a ising om he me al elec odes hemsel es, wi h he s onges lines iden i ied a 324.8 and
327.4 nm in he UV egion; 510.6, 515.3 and 521.8 nm in he isible egion and 793.3 and 809.3 nm in
he NIR egion (see Figu es 6and 7).
Wa e 2019, 11, x FOR PEER REVIEW 6 o 17
Figu e 5. Su ey spec um o plasma in he liquid/ca i a ion bubble cloud en i onmen .
The s onges emission in he UV spec al egion came om OH adicals (Figu e 6), wi h he
s onges emission o he OH 2Σ→2Π g ound s a e sys em occu ing a 306 nm (bands 0-0 and 1-1)
and a weake emission a 281 nm (band 1-0). Unde isible and NIR, he spec um also showed s ong
a omic hyd ogen lines (Balme se ies; Hα a 656 nm, Hβ a 486 nm and Hγ a 434 nm) and a omic
oxygen lines (3s-3p iple s a 777.4 nm, 5S*-5P and 844.6 nm, 3S*-3P). The p esence o a wa e laye
be ween he discha ge and he PMMA ube con ibu ed o abso p ion o he discha ge emission [22].
Fo example, ni ogen adical molecules p esen in he discha ge a mosphe e we e dilu ed, as shown
by a compa ison be ween he emission o he second posi i e N2 sys em (SPS; C 3Π→B 3Π) wi h he
i s nega i e N2+ ion sys em (FNS; 2Σ→2Σ, g ound s a e). The SPS comp ised nume ous ib a ion
bands (e.g., band 0-0 a 337.1 nm), while he FNS spec um only showed a weak band a 391.4 nm
(band 0-0). Based on he second posi i e ni ogen sys em spec um, he o a ional empe a u e o he
N2 molecules was es ima ed a 3600 ± 300 K.
The emission spec a, showing a omic oxygen, a omic hyd ogen and exci ed ni ogen
molecules/ions in he discha ge, clea ly indica e ha wa e apou molecules we e dissocia ed o OH
adicals. The p esence o exci ed ni ogen molecules and a omic oxygen also sugges s he p esence o
ni ogen oxide species [23], commonly obse ed in discha ge spec a o discha ges used o p oduce
RONS in wa e [24,25]. While o he spec oscopic me hods (e.g., Fou ie ans o m in a ed
abso p ion, lase abso p ion, elec on pa amagne ic esonance), along wi h luo escence/colo ime ic
measu emen s [22,26,27], may be be e placed o con i m and quan i y RONS, ou own spec oscopic
e alua ion clea ly showed p oduc ion o RONS by HC + plasma.
The spec um also consis s o a omic coppe lines (see Figu es 6 and 7) a ising om he ma e ial
o me al elec odes. The s onges iden i ied lines we e a 324.8 and 327.4 nm in he UV egion, 510.6,
515.3 and 521.8 nm in he isible egion and 793.3 and 809.3 nm in he NIR egion.
Fo he iden i ica ion o he spec al lines and bands he ollowing li e a u e was used: [19–21]
Figu e 5. Su ey spec um o plasma in he liquid/ca i a ion bubble cloud en i onmen .
The s onges emission in he UV spec al egion came om OH adicals (Figu e 6), wi h he
s onges emission o he OH
2Σ→2Π
g ound s a e sys em occu ing a 306 nm (bands 0-0 and 1-1)
and a weake emission a 281 nm (band 1-0). Unde isible and NIR, he spec um also showed s ong
a omic hyd ogen lines (Balme se ies; H
α
a 656 nm, H
β
a 486 nm and H
γ
a 434 nm) and a omic

Wa e 2020,12, 8 6 o 14
oxygen lines (3s-3p iple s a 777.4 nm, 5S
*
-5P and 844.6 nm, 3S
*
-3P). The p esence o a wa e laye
be ween he discha ge and he PMMA ube con ibu ed o abso p ion o he discha ge emission [
22
].
Fo example, ni ogen adical molecules p esen in he discha ge a mosphe e we e dilu ed, as shown
by a compa ison be ween he emission o he second posi i e N
2
sys em (SPS; C
3Π→
B
3Π
) wi h he
i s nega i e N
2+
ion sys em (FNS;
2Σ→2Σ
, g ound s a e). The SPS comp ised nume ous ib a ion
bands (e.g., band 0-0 a 337.1 nm), while he FNS spec um only showed a weak band a 391.4 nm
(band 0-0). Based on he second posi i e ni ogen sys em spec um, he o a ional empe a u e o he
N2molecules was es ima ed a 3600 ±300 K.
Wa e 2019, 11, x FOR PEER REVIEW 7 o 17
Figu e 6. Nea UV/VIS spec um o plasma in he liquid/ca i a ion bubble cloud en i onmen .
Figu e 7. VIS/NIR spec um o plasma in he liquid/ca i a ion bubble cloud en i onmen .
3.2. Mic ocys is G ow h Inhibi ion unde Op imal Condi ions wi h Nu ien En ichmen
We unde ook an ini ial 8-day expe imen al ial, wi h samples aken a e one, h ee and i e
ea men cycles, in o de o assess he numbe o cycles needed o (a) p omo e Mic ocys is damage,
and (b) assess whe he cyanobac e ia a e able o eco e g ow h and me abolic ac i i y ollowing HC
+ plasma ea men unde op imal condi ions. This i s es con i med h ee cycles as being su icien
o inhibi cyanobac e ial g ow h unde bo h HC and HC + plasma ea men s, wi h he esul s o HC
+ plasma e en mo e p onounced han o HC alone (Figu es 8 and 9). While g ow h o HC- ea ed
Mic ocys is began o eco e one week a e a single cycle o ea men (Figu e 8), i emained s ongly
inhibi ed ollowing ea men by HC + plasma, wi h li le me abolic ac i i y e iden a e eigh days
unde op imal cul i a ion condi ions (Figu e 9).
Figu e 6. Nea UV/VIS spec um o plasma in he liquid/ca i a ion bubble cloud en i onmen .
Wa e 2019, 11, x FOR PEER REVIEW 7 o 17
Figu e 6. Nea UV/VIS spec um o plasma in he liquid/ca i a ion bubble cloud en i onmen .
Figu e 7. VIS/NIR spec um o plasma in he liquid/ca i a ion bubble cloud en i onmen .
3.2. Mic ocys is G ow h Inhibi ion unde Op imal Condi ions wi h Nu ien En ichmen
We unde ook an ini ial 8-day expe imen al ial, wi h samples aken a e one, h ee and i e
ea men cycles, in o de o assess he numbe o cycles needed o (a) p omo e Mic ocys is damage,
and (b) assess whe he cyanobac e ia a e able o eco e g ow h and me abolic ac i i y ollowing HC
+ plasma ea men unde op imal condi ions. This i s es con i med h ee cycles as being su icien
o inhibi cyanobac e ial g ow h unde bo h HC and HC + plasma ea men s, wi h he esul s o HC
+ plasma e en mo e p onounced han o HC alone (Figu es 8 and 9). While g ow h o HC- ea ed
Mic ocys is began o eco e one week a e a single cycle o ea men (Figu e 8), i emained s ongly
inhibi ed ollowing ea men by HC + plasma, wi h li le me abolic ac i i y e iden a e eigh days
unde op imal cul i a ion condi ions (Figu e 9).
Figu e 7. VIS/NIR spec um o plasma in he liquid/ca i a ion bubble cloud en i onmen .
The emission spec a, showing a omic oxygen, a omic hyd ogen and exci ed ni ogen
molecules/ions in he discha ge, clea ly indica e ha wa e apou molecules we e dissocia ed
o OH adicals. The p esence o exci ed ni ogen molecules and a omic oxygen also sugges s he
p esence o ni ogen oxide species [
23
], commonly obse ed in discha ge spec a o discha ges used o
p oduce RONS in wa e [
24
,
25
]. While o he spec oscopic me hods (e.g., Fou ie ans o m in a ed
abso p ion, lase abso p ion, elec on pa amagne ic esonance), along wi h luo escence/colo ime ic
measu emen s [
22
,
26
,
27
], may be be e placed o con i m and quan i y RONS, ou own spec oscopic
e alua ion clea ly showed p oduc ion o RONS by HC +plasma.
The spec um also consis s o a omic coppe lines (see Figu es 6and 7) a ising om he ma e ial
o me al elec odes. The s onges iden i ied lines we e a 324.8 and 327.4 nm in he UV egion, 510.6,
515.3 and 521.8 nm in he isible egion and 793.3 and 809.3 nm in he NIR egion.
Fo he iden i ica ion o he spec al lines and bands he ollowing li e a u e was used: [19–21]
Wa e 2020,12, 8 7 o 14
3.2. Mic ocys is G ow h Inhibi ion unde Op imal Condi ions wi h Nu ien En ichmen
We unde ook an ini ial 8-day expe imen al ial, wi h samples aken a e one, h ee and i e
ea men cycles, in o de o assess he numbe o cycles needed o (a) p omo e Mic ocys is damage,
and (b) assess whe he cyanobac e ia a e able o eco e g ow h and me abolic ac i i y ollowing HC +
plasma ea men unde op imal condi ions. This i s es con i med h ee cycles as being su icien o
inhibi cyanobac e ial g ow h unde bo h HC and HC +plasma ea men s, wi h he esul s o HC
+plasma e en mo e p onounced han o HC alone (Figu es 8and 9). While g ow h o HC- ea ed
Mic ocys is began o eco e one week a e a single cycle o ea men (Figu e 8), i emained s ongly
inhibi ed ollowing ea men by HC +plasma, wi h li le me abolic ac i i y e iden a e eigh days
unde op imal cul i a ion condi ions (Figu e 9).
Wa e 2019, 11, x FOR PEER REVIEW 8 o 17
Figu e 8. G ow h o a Mic ocys is cul u e ollowing HC ea men . RFU –Rela i e luo escence uni s.
Figu e 9. G ow h o a Mic ocys is cul u e ollowing HC + plasma ea men .
3.3. Mechanisms o Mic ocys is Biomass Remo al
Based on he esul s o he i s expe imen al ial, we decided on h ee cycles as he op imal
ea men egime. In he second expe imen al ial, he e o e, cyanobac e ial ea men samples we e
aken a e one, wo and h ee cycles.
We included he measu emen o chlo ophyll-a o ou expe imen al design in o de o con i m
quan i a i e emo al o cyanobac e ial biomass om he wa e column. The esul s ended o mi o
hose om he g ow h inhibi ion es , wi h a mo e apid dec ease in chlo ophyll concen a ion
ollowing wo o mo e cycles o HC + plasma ea men compa ed wi h HC ea men alone (Figu es
10 and 11).
Cyanobac e ial biomass only showed a slow decline ollowing ea men by HC alone, wi h a
no iceable e ec only de ec able a e 3–6 days (Figu e 10). In compa ison, ea men by HC + plasma
Figu e 8. G ow h o a Mic ocys is cul u e ollowing HC ea men . RFU –Rela i e luo escence uni s.
Wa e 2019, 11, x FOR PEER REVIEW 8 o 17
Figu e 8. G ow h o a Mic ocys is cul u e ollowing HC ea men . RFU –Rela i e luo escence uni s.
Figu e 9. G ow h o a Mic ocys is cul u e ollowing HC + plasma ea men .
3.3. Mechanisms o Mic ocys is Biomass Remo al
Based on he esul s o he i s expe imen al ial, we decided on h ee cycles as he op imal
ea men egime. In he second expe imen al ial, he e o e, cyanobac e ial ea men samples we e
aken a e one, wo and h ee cycles.
We included he measu emen o chlo ophyll-a o ou expe imen al design in o de o con i m
quan i a i e emo al o cyanobac e ial biomass om he wa e column. The esul s ended o mi o
hose om he g ow h inhibi ion es , wi h a mo e apid dec ease in chlo ophyll concen a ion
ollowing wo o mo e cycles o HC + plasma ea men compa ed wi h HC ea men alone (Figu es
10 and 11).
Cyanobac e ial biomass only showed a slow decline ollowing ea men by HC alone, wi h a
no iceable e ec only de ec able a e 3–6 days (Figu e 10). In compa ison, ea men by HC + plasma
Figu e 9. G ow h o a Mic ocys is cul u e ollowing HC +plasma ea men .
3.3. Mechanisms o Mic ocys is Biomass Remo al
Based on he esul s o he i s expe imen al ial, we decided on h ee cycles as he op imal
ea men egime. In he second expe imen al ial, he e o e, cyanobac e ial ea men samples we e
aken a e one, wo and h ee cycles.
We included he measu emen o chlo ophyll-a o ou expe imen al design in o de o con i m
quan i a i e emo al o cyanobac e ial biomass om he wa e column. The esul s ended o mi o
hose om he g ow h inhibi ion es , wi h a mo e apid dec ease in chlo ophyll concen a ion ollowing
wo o mo e cycles o HC +plasma ea men compa ed wi h HC ea men alone (Figu es 10 and 11).
Wa e 2020,12, 8 8 o 14
Wa e 2019, 11, x FOR PEER REVIEW 9 o 17
esul ed in a 95% d op in cyanobac e ial biomass wi hin a single day (Figu e 11). Mic oscopic
examina ion con i med ha HC ea men a ec ed cell s uc u e, he cellula su ace and, especially,
he gas esicles and subcellula s uc u e ha allow Mic ocys is o loa . This was con i med by
mic oscopic obse a ion, which showed abundan gas esicles (black do s inside cells) in con ol cells
(Figu e 12A) bu a la ge numbe o emp y cells a e jus one ca i a ion cycle (Figu e 12B).
Hyd odynamic ca i a ion can be used in wa e ea men echnologies o di e en pu poses ( o
e iew, see [28]) and can be combined wi h o he echnologies like ad anced oxygena ion
echnologies, e c. He e, we combined HC wi h plasma and, in compa ison o HC use only, all gas
esicles had collapsed in e e y cell ollowing he combina ion HC + plasma ea men (Figu e 12C).
Howe e , di e en mechanisms we e employed when we combined HC wi h plasma. As a esul ,
Mic ocys is biomass was almos comple ely emo ed om he wa e column a e one day o
ea men , and we hypo hesize ha he cells had insu icien ene gy o main ain me abolic ac i i y.
This hypo hesis can be p o en by measu emen o pho osyn he ic ac i i y.
Figu e 10. Remo al o cyanobac e ial biomass ollowing HC ea men .
Figu e 10. Remo al o cyanobac e ial biomass ollowing HC ea men .
Wa e 2019, 11, x FOR PEER REVIEW 10 o 17
Figu e 11. Remo al o cyanobac e ial biomass ollowing HC + plasma ea men .
(A)
Figu e 11. Remo al o cyanobac e ial biomass ollowing HC +plasma ea men .
Cyanobac e ial biomass only showed a slow decline ollowing ea men by HC alone, wi h a
no iceable e ec only de ec able a e 3–6 days (Figu e 10). In compa ison, ea men by HC +plasma
esul ed in a 95% d op in cyanobac e ial biomass wi hin a single day (Figu e 11). Mic oscopic
examina ion con i med ha HC ea men a ec ed cell s uc u e, he cellula su ace and, especially,
he gas esicles and subcellula s uc u e ha allow Mic ocys is o loa . This was con i med by
mic oscopic obse a ion, which showed abundan gas esicles (black do s inside cells) in con ol cells
(Figu e 12A) bu ala genumbe o emp ycellsa e jus one ca i a ion cycle(Figu e12B). Hyd odynamic
ca i a ion can be used in wa e ea men echnologies o di e en pu poses ( o e iew, see [
28
])
and can be combined wi h o he echnologies like ad anced oxygena ion echnologies, e c. He e, we
combined HC wi h plasma and, in compa ison o HC use only, all gas esicles had collapsed in e e y
cell ollowing he combina ion HC +plasma ea men (Figu e 12C). Howe e , di e en mechanisms
we e employed when we combined HC wi h plasma. As a esul , Mic ocys is biomass was almos
comple ely emo ed om he wa e column a e one day o ea men , and we hypo hesize ha
he cells had insu icien ene gy o main ain me abolic ac i i y. This hypo hesis can be p o en by
measu emen o pho osyn he ic ac i i y.
Wa e 2020,12, 8 9 o 14
Wa e 2019, 11, x FOR PEER REVIEW 10 o 17
Figu e 11. Remo al o cyanobac e ial biomass ollowing HC + plasma ea men .
(A)
Wa e 2019, 11, x FOR PEER REVIEW 11 o 17
(B)
(C)
Figu e 12. (A) Abundan gas esicles (black do s inside cells) in con ol Mic ocys is cells; (B) Mic ocys is
cells a e he i s HC ca i a ion cycle. Red a ows show he cells wi h collapsed gas esicles; (C)
Mic ocys is cells a e HC + plasma ea men , indica ing in ac cells wi h collapsed gas esicles.
Cyanobac e ial pho osyn he ic ac i i y, measu ed using dynamic o chlo ophyll luo escence
(ChlFl) cha ac e is ics, exhibi ed signi ican changes al eady a e 24 h a e HC ea men , wi h he
e ec s pa icula ly no iceable ollowing ea men wi h HC + plasma (Figu e 13). Compa ed o he
un ea ed con ol, basal ChlFl (F0) alues inc eased in samples exposed o h ee cycles o HC and HC
+ plasma (Figu e 13A). A conside able d op in F0 alues was obse ed 48 and 72 h a e HC + plasma
Figu e 12.
(
A
) Abundan gas esicles (black do s inside cells) in con ol Mic ocys is cells; (
B
)Mic ocys is
cells a e he i s HC ca i a ion cycle. Red a ows show he cells wi h collapsed gas esicles;
(C)Mic ocys is cells a e HC +plasma ea men , indica ing in ac cells wi h collapsed gas esicles.